Simultaneous quantification of nucleic acids in diseased cells

ABSTRACT

Processes and methods for the simultaneous quantification of nucleic acids in diseased cells that are based on real-time PCR are provided. The real-time PCR protocol is an excellent tool for reliable quantification of in vitro drug screening and evaluation protocols to determine the efficacy of potential anti-viral agents. Quantification using these simultaneous PCR cycle threshold (Ct) detection techniques during one-step real-time RT-PCR (Applied Biosystems, CA) eliminates the variability resulting from quantification of end-point RT-PCR products. In addition, the mitochondrial toxicity assay is an added tool to assess potential side-effects for these chemotherapeutic agents.

This application claims priority to U.S. Provisinal Application No.60/241,488, filed on Oct. 18, 2000, U.S. Provisinal Application No.60/256,067 filed on Dec. 15, 2000 and U.S. Provisinal Application No.60/282,156, filed on Apr. 6, 2001.

FIELD OF THE INVENTION

This application is in the area of processes for the detection andanalysis of viral infections and mitochondrial toxicity, and forprocesses for the identification of active compounds for the treatmentof viral infections and processes to measure mitochondrial toxicityresulting from drug therapies.

BACKGROUND OF THE INVENTION

The detection and quantification of nucleic acid sequences is ofimportance for a wide range of applications. The most widely used methodto detect nucleic acids are based on the polymerase chain reaction(PCR). PCR is used to amplify a segment of DNA flanked by stretches ofknown sequences. Two oligonucleotides binding to these known flankingsequences are used as primers for a series of in vitro reactions thatare catalyzed by a DNA polymerase. These oligonucleotides typically havedifferent sequences and are complementary to sequences that lie onopposite strands of the template DNA and flank the segment of DNA thatis to be amplified. The template DNA is first denatured by heat in thepresence of a large molar excess of each of the two oligonucleotides andthe four 2′-deoxynucleotide triphosphates. The reaction mixture is thencooled to a temperature that allows the oligonucleotide primers toanneal to their target sequences. Afterwards, the annealed primers areextended by the DNA polymerase. The cycle of denaturation, annealing,and DNA-synthesis is then repeated about 10 to 50 times. Since theproducts of one cycle are used as a template for the next cycle theamount of the amplified DNA fragment is theoretically doubled with eachcycle resulting in a PCR-efficiency of 100%.

“Real-time PCR” refers to a polymerase chain reaction that is monitored,usually by fluorescence, over time during the amplification process, tomeasure a parameter related to the extent of amplification of aparticular sequence, such as the extent of hybridization of a probe toamplified target sequences. The DNA generated within a PCR is detectedon a cycle by cycle basis during the PCR reaction. The amount of DNAincreases faster the more template sequences are present in the originalsample. When enough amplification products are made a threshhold isreached at which the PCR products are detected. Thus amplification anddetection are performed simultaneously in the same tube.

In biological research, PCR has accelerated the study of testing forcommunicable diseases. Medical applications of PCR include identifyingviruses, bacteria and cancerous cells in human tissues. PCR can even beused within single cells, in a procedure called in situ (in-site) PCR,to identify specific cell types. PCR can also be applied to theamplification of RNA, a process referred to as reverse transcriptase PCR(RT-PCR). RT-PCR is similar to regular PCR, with the addition of aninitial step in which DNA is synthesized from the RNA target using anenzyme called a reverse transcriptase. A wide variety of RNA moleculeshave been used in RT-PCR, including ribosomal RNA, messenger RNA andgenomic viral RNA.

PCR itself is quite simple, but sample preparation can be laborious. Thegoals of sample preparation include the release of nucleic acid (DNA orRNA), concentration of the nucleic acid to a small volume for PCR, andremoval of inhibitors of PCR. Inhibitors of PCR are naturally occurringsubstances which reduce the efficiency of PCR, and which are oftenpresent in clinical samples. When the specimen contains a large amountof target nucleic acid, sample preparation is trivial. But samplepreparation is more difficult in most clinical specimens, particularlywhen a large volume specimen must be processed and only a few pathogensare present. Complex protocols are often required.

Since PCR detects the presence or absence of a particular nucleic acidtarget, it will only detect a pathogen if its nucleic acid is present inthe particular specimen. PCR detects nucleic acids from living or deadmicrobes. This must be recognized if PCR is used to monitor response totherapy. PCR provides at most nucleic acid sequence information. PCR canbe used to screen for drug resistance mutations, but it does not providedirect antibiotic susceptibility data.

Appropriate controls are necessary when PCR is used diagnostically.These include negative controls, positive controls and specificitycontrols. Negative controls (no target DNA) are needed to detectcontamination. Contamination can occur during sample preparation orreagent mixing, so negative controls need to be processed in parallelwith clinical samples. Negative controls should be interspersed amongthe samples to detect cross-contamination from sample to sample.Contamination is frequently intermittent; a sufficient number ofnegative controls must be included to detect low rates of contamination.Most published studies have not included a sufficient number of negativecontrols.

Positive controls include a small number of target DNA copies. Positivecontrols are needed to ensure efficient release of target DNA frompathogens, to guard against loss of DNA during sample processing, and toidentify the presence of inhibitors (natural substances sometimespresent in clinical samples that reduce PCR efficiency). Positivecontrols should be processed in parallel with clinical specimens.Clinical specimens vary in the presence of inhibitors of PCR, and it maybe necessary to add an internal positive control for each sample. Theinternal positive controls have the same recognition sites as the targetDNA, but are designed with some difference in the internal sequence.Amplification of the internal positive controls can be distinguishedfrom that of the real target DNA.

Specificity controls are needed to determine the range of target DNAsthat will be amplified by the PCR assay. For assays designed to detectpathogens in clinical samples, human DNA samples must be tested toensure that the PCR primers do not recognize a human DNA target bychance. Related pathogens must be tested to determine the range ofspecies/strains that will be amplified. Specificity controls are neededonly once, when a new PCR assay is designed. Negative and positivecontrols must be included every time samples are processed, and shouldbe processed simultaneously with the clinical samples.

PCR has been used in three broad categories of diagnostic procedures,namely detection, characterization and quantification.

Detection is the most difficult PCR procedure, especially when thenumber of pathogens in the specimen is low. The PCR must be conductedunder conditions of high sensitivity. Many temperature cycles are used,or a nested protocol is used in which the products from the firstreaction are re-amplified with a second set of primers. This makes PCRfor detection especially prone to carryover contamination. Samplepreparation may be laborious, as there is an attempt to process as largea specimen volume as possible. Inhibitors of PCR occur naturally in manyclinical samples, and are a major limitation. Numerous positive andnegative controls must be included as described above.

In a characterization procedure, nucleic acid variants are identifiedbased on the nucleic acid sequence between the two PCR primers. Manytechniques can be used to detect variable sequences, including lengthpolymorphism, changes in restriction sites, and direct DNA sequencing.This is often the easiest type of PCR to carry out clinically. Amplequantities of nucleic acid target can be present in the specimen, eitheran already grown bacterial or viral culture or a clinical sample withlarge numbers of microbes. Goals can include rapid detection of drugresistance mutations, assignment of strains to clinically meaningfulphylogenetic groups, or epidemiological tracing.

Quantitation (indicating how many copies of the target nucleic acid arepresent) has primarily been applied to chronic viral infections,especially hepatitis C virus (HCV) and human immunodeficiency virus(HIV) infections. The level of viremia has prognostic implications, andhas been used to demonstrate response to antiviral drugs. PCR is quitesensitive, but it is not inherently quantitative. The amount of thefinal PCR product is usually similar from an initial sample containing10 or 10,000 copies. This limitation can be overcome by serial dilutionof the clinical sample until no target DNA is detected, or by theaddition of synthetic competitor DNA molecules. The competitor moleculeshave regions complementary to the two primers, but differ in some wayfrom the natural target (e.g., a different length). By comparing theamount of the natural and competitor PCR products, a rough estimation ofthe number of target molecules in the sample is possible.

PCR has been applied in the research setting to hundreds of pathogens,and has yielded important insights into pathogenesis and epidemiology ofmany infectious diseases. For clinical purposes, PCR-based diagnostictests are best applied when the following conditions are fulfilled: (1)The results of the test will make a clear clinical difference and atherapy will be given or withheld based on the results of PCR; (2)routine culture methods are limited because the microbe cannot be grown(e.g., Mycobacterium leprae, HCV), grows slowly (e.g., M. tuberculosis),or is difficult to culture (e.g., Brucella species, HIV); and (3) thereis an accessible clinical specimen which contains large numbers ofmicrobes (e.g., blood for HCV or HIV).

PCR has been useful in a variety of chronic virus infections (HIV, HCV,hepatitis B virus, human papillomavirus and cytomegalovirus). PCR hasbeen crucial for the detection of HIV infection in neonates, sincematernal antibodies complicate serologic diagnosis. Quantitation of HIVand HCV viremia by PCR has important prognostic implications, and hasbeen used to monitor response to drug therapy. PCR is useful for therapid diagnosis of pulmonary infections in immuno-compromised hosts,particularly for cytomegalovirus and Pneumocystis carinii.

HIV

The human immunodeficiency virus type-1 (HIV-1) is a retrovirusbelonging to the family of the Lentiviridae. One of the characteristicfeatures of this virus group is that the members replicate over a DNAintermediate through the viral encoded reverse transcriptase (RT) enzymeactivity. The high replication rate combined with the low fidelity ofthat reverse transcriptase enzyme provides the virus with an extremelyhigh genomic flexibility. As a consequence, different levels of geneticvariability are observed for HIV-1. The epidemic is characterized by thepresence of clades within the M-group virus, but there is also anO-group and an N-group virus described, each of them again harboring avariety of clades. Quasispecies populations within the infectedindividual are also seen. Clinically, there are some importantconsequences to this quasispecies concept, for example, in vaccinedevelopment and immune escape. This concept contributes to the emergenceof drug resistant variants that surface under antiviral treatments.

In order to control the course of the disease in infected individuals,potent highly active anti-retroviral therapies (HAART) have beendesigned. Due to the ongoing replication of the virus, anti-retroviraldrug resistance eventually develops, leading to therapy failure.Therefore, there is an ongoing need for more and more potent anti-HIV-1drugs.

To assess the efficacy of drugs in the treatment of patients in vivo,clinical markers of virus replication needed to be defined. In the past,some surrogate markers, like CD-cell count, have been used. Morerecently, some commercial assays like Quantiplex (Chiron), NucliSense(Organon-Teknika) and Amplicor HIV-1 Monitor (Roche) were developed todirectly measure viral load. These viral load determinations proved tobe an excellent tool in monitoring therapeutic efficiency for HAART andfor clinical trials with new experimental drugs.

The design of an HIV-1 viral load test is a real challenge. Ideally, aviral load test should fulfill to the following criteria:

-   -   i) be able to detect the huge variability of clades within one        group with the same efficiency;    -   ii) have a dynamic range of at least five logs or higher; and    -   iii) the lower limit of detection should be as low as a few        viral copies/mL. Although variability at the PCR-primer binding        sites is a real concern in assay development, RT-PCR based        assays are considered as the most sensitive technologies.        Mitochondrial Toxicity

Mitochondrial toxicity is clearly recognized as an adverse effect oflong-term use of antiviral agents, in particular reverse transcriptaseinhibitors. Clinical features of this mitochondrial toxicity varydepending on the tissues that are affected. It is largely dependent onthe aerobic metabolism needed for energy supply required for thatparticular tissue. Most toxic events are reversible at an early stage,however lactic acidosis is often irreversible and can result in death.

The common pathway of antiviral agent induced toxicity is mitochondrialdysfunction. The antiviral agent (most likely the triphosphate form of anucleoside analogue) inhibits the mitochondrial DNA polymerase γ leadingto the loss of mitochondria. This enzyme is essential for thereplication of the mitochondrial genome. Tissues with high ATP demandare most susceptible to mitochondrial toxicity.

The mechanism underlying this mitochondrial dysfunction includes failureof energy dependent ionic balance. Subsequently, there is an increase inintracellular calcium, initiating lipolysis and proteolysis, and leadingto the accumulation of lactic acid and partial reduction of therespiratory activities.

Since the mitochondrial dysfunction develops over months and symptomsare initially mild, it is important to develop sensitive diagnostictests that allow determination of the enzyme activity and inhibition bythe selected antiviral agent. Evenly important, new candidate antiviralagents need to be evaluated for their unfavorable DNA polymerase γinhibiting capacities.

Hepatitis C

Hepatitis C virus (HCV) infection is a pandemic infection, and is amajor cause of liver disease. Reports of successful treatment of HCVinfection with interferon have increased interest in applications ofRT-PCR.

Available tests for HCV infection are limited. Initial serologic testsfor HCV had poor sensitivity. Second and third-generation serologictests have improved sensitivity, but are still not completelydependable. HCV RNA is readily detected in serum using RT-PCR. Viremicpatients typically have very high viral titers.

PCR has been applied to the diagnosis of HCV infection in a variety ofclinical settings. HCV can be detected as early as one week afterinfection, and PCR can be used to detect HCV infection during the“window” period between infection and seroconversion. HCV PCR is usefulfor detecting HCV in seronegative individuals with liver disease. It canbe used to confirm maternal to fetal spread of HCV. HCV PCR may beuseful in the evaluation of seropositive individuals as candidates forinterferon or other therapies. Portions of HCV-seropositive patients arenegative by HCV PCR, and may have resolved their infections.PCR-negative individuals have lower serum transaminase concentrationsand less histologic activity on liver biopsies. Long-term follow-upstudies are needed, but it may be reasonable to withhold therapy frompatients with negative HCV PCR results.

The amount of HCV viremia can be determined by either quantitative PCR.PCR is sensitive and is quantitative over a wide range of viral titers.High-titer viremia is correlated with an advanced disease stage. Theprognostic value of HCV quantitation awaits prospective studies, but thelevel of viremia may be useful in selecting candidates for therapy.Quantitative HCV PCR also appears to be useful in monitoring theresponse to therapy.

WO 00/44936 filed by Bavarian Nordic Research Institute A/S describes areal-time PCR method for the detection and quantification of variants ofnucleic acid sequences which differ in the probe-binding site. Themethod is based in the complete or partial amplification of the sameregion of the variants and the addition of two or more oligonucleoitdeprobes to the same PCR mixture, each probe being specific for theprobe-binding site of at least one variant.

WO 01/66799 filed by E.I. DuPont Nemours and Company discloses aPCR-based dsDNA quantification method that monitors the fluorescence ofa target, whose melting characteristics is predetermined, during eachamplification cycle at selected time points. By selecting targets withdistinguishing melting curve characteristics, multiple targets can besimultaneously detected.

WO 00/68436 filed by Nationales Zentrum fur Retroviren disclosessequences allowing the detection and quantification of humanimmunodeficiency virus.

U.S. Pat. No. 6,235,504 assigned to the Rockefeller University describesmethods for identifying genetic sequences useful as genomic equivalentmarkers for organisms.

U.S. Pat. No. 6,210,875 discloses a process for determining the efficacyof antiviral therapy in an HIV-infected host that includes detecting thelevel of transcriptionally active HIV in the monocytes of the subject ata plurality of times by simultaneously exposing the monocytes to anoligonucleotide probe that specifically binds to at least a portion ofHIV mRNA and exposing the monocytes to an antibody, wherein theoligonucleotide probe is labeled with a fluorescent label, comparing thedetected HIV levels, and correlating the HIV levels over time with thetherapy regimen.

U.S. Pat. No. 5,843,640 discloses an in situ process of simultaneouslydetecting a specific predetermined nucleic acid sequence and a specificpredetermined cellular antigen in the same cell.

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Although assays exist for the diagnosis and evaluation of viralinfections, additional assays and kits are needed that provide a moresensitive or precise analysis of the condition of a diseased cell. Moresensitive and precise methods are also needed to assess the activity ofa compound or substance against a target virus and to assess hosttoxicity induced by the compound or substance.

It is therefore an object of the present invention to provide a processfor the identification of active compounds for the treatment of viralinfections.

It is another object of the present invention to provide a process tomeasure mitochondrial toxicity.

It is another object of the present invention to provide a process forthe detection and analysis of viral infections.

It is a further object of the invention to provide a process for thedetection and analysis of mitochondrial toxicity.

SUMMARY OF THE INVENTION

Processes and methods for the simultaneous quantification of nucleicacids in diseased cells that are based on real-time PCR are provided.The real-time-PCR protocol is an excellent tool for reliablequantification of in vitro drug screening and evaluation protocols todetermine the efficacy of potential anti-viral agents. Quantificationusing these simulateous PCR cycle threshold (Ct) detection techniquesduring one-step real-time RT-PCR (Applied Biosystems, CA) eliminates thevariability resulting from quantification of end-point RT-PCR products.In addition, the mitochondrial toxicity assay is an added tool to assesspotential side-effects for these chemotherapeutic agents.

This real time multiplex PCR system includes the simultaneousmeasurements of cellular DNA (for example rDNA) or cellular RNA (forexample rRNA or β-actin m-RNA), and viral RNA or DNA. In one embodiment,the simultaneous real time analysis of host and viral nucleic acidallows the calculation of a sensitivity assay that indicates thecomparative condition of the host cell and the virus. In a separateaspect of the invention, multiplex PCR is used to simultaneously measurethe nuclear and the mitochondrial nucleic acid of a cell to provideinformation on drug toxicity, or to evaluate a cell (in vivo or invitro) that may exhibit a disease that involves mitochondrial toxicity,such as peripheral neuropathy, peripheral lipodystrophy, or a geneticdisease that causes a disruption in mitochondrial DNA or RNA synthesis.

The methods and processes are economic, non-radioactive, rapid,accurate, reproducible, and amenable to large through-put. It canprovide a dynamic range of quantification with linearity of over 5-7logs. One way to express the antiviral effectiveness of a compound is tosubtract the threshold RT-PCR cycle of the test compound with theaverage threshold RT-PCR cycle of the negative control. This value iscalled DeltaCt (ΔCt). A ΔCt of 3.3 equals a 1-log reduction (equalsEC₉₀) in viral nucleic acid production. Compounds that result in areduction of viral nucleic acid greater than 1.5, or more preferred, 2Ct values (75% reduction of viral nucleic acid) are typically usefulcompounds for the inhibition of viral growth.

With the availability of both the viral ΔCt data and the host ΔCt, aspecificity parameter can be introduced. This parameter is obtained bysubtracting the host ΔCt value from the viral ΔCt value. This results inΔΔCt values; a value above 0 means that there is more inhibitory effecton the viral nucleic acid, a ΔΔCt value below 0 means that the hostnucleic acid is more affected. As a general rule, ΔΔCt values above 2are considered as significantly different from the no-drug treatmentcontrol, and hence, exhibits useful antiviral activity. However,compounds with a ΔΔCt value of less than 2, but showing limitedmolecular cytotoxicty data (rRNA ΔCT between 0 and 2) may also bedesired for certain applications requiring compounds with low toxicity.

As an example, a compound might reduce the host RNA polymerase activity,but not the host DNA polymerase activity. Therefore, quantification ofrDNA or β-actin DNA (or any other host DNA fragment) and comparison withDNA levels of the no-drug control is a relative measurement of theinhibitory effect of the test compound on cellular DNA polymerases. Withthe availability of both the HCV ΔCt data and the rDNA ΔCt, aspecificity parameter can be introduced. This parameter is obtained bysubtracting both ΔCt values from each other. This results in ΔΔCtvalues; a value above 0 means that there is more inhibitory effect onthe viral encoded polymerase, a ΔΔCt value below 0 means that the hostrDNA levels are more affected than the viral nucleic acid levels. As ageneral rule, ΔΔCt values above 2 are considered as significantlydifferent from the no-drug treatment control, and hence, is aninterested compound for further evaluation. However, compounds with aΔΔCt value of less than 2, but with limited molecular cytotoxicty (rDNAΔCT between 0 and 2) are also possible active candidate compounds forfurther evaluation

In a first embodiment, a process for assessing a viral disease isprovided that includes contacting nucleic acid from a viral infectedhost cell with an amplification reaction mixture that contains at leasttwo primers and/or probes that provide detectable signals during apolymerase chain reaction, wherein

-   -   the first primer and/or probe provides a detectable signal on        the occurrence on the transcription of host nucleic acid; and    -   the second primer and/or probe provides a second detectable        signal on the occurrence on the transcription of viral nucleic        acid.

In a particular embodiment, the level of transcription of the viral andhost nucleic acid is compared to that of a standard, including but notlimited to, a known viral infected host cell, or alternatively, aninternal standard can be established by comparing the extent oftranscription of the host and viral nucleic acid over a number ofsamples from the host to monitor and measure the change in infection. Inanother embodiment, the data can be assessed as described above throughthe use of ΔCT and ΔΔCt values.

In a preferred embodiment, the nucleic acid is a consensus or non-codingsequence, which can be either 5′ or 3′ to the target expressed sequence.In one embodiment, the non-coding sequence is an intron or a partthereof. Non-limiting examples are non-coding sequences from β-actin orGAPDH.

The host nucleic acid can be nuclear or cytoplasmic, and in particular,mitochondrial nucleic acid, and the viral nucleic acid can be either DNAor RNA.

This process can be used to evaluate the ability of the compound orsubstance to inhibit the replication of any virus, including but notlimited to a virus from the Retroviridae, Flaviviridae,Orthomyxoviridae, Paramyxoviridae, Herpesviridae, Hepadnaviridae,Picornaviridae, Reoviridae, Poxyiridae, Adenoviridae, Papoviridae,Parvoviridae, Bunyaviridae, Filoviridae, Arenaviridae or Togaviridaefamily. In particular, the virus is HIV, hepatitis (including but notlimited to A, B, C, D and G), BVDV (bovine diarrhea virus), herpessimplex, Adenovirus type 1, influenza, including influenza A (HINI),influenza A (H3N2), influenza B, influenza C and influenza D, measles,mumps, parainfluenza type 3, RSV (respiratory syncytial virus), HSV(herpes simplex virus), EBV (Epstein Barr virus), CMV (cytomegalovirus)or West Nile Virus.

In a second embodiment, a process for assessing a disease state thatincludes a disruption in mitochondrial DNA or RNA synthesis is providedthat includes contacting nucleic acid from a host with an amplificationreaction mixture that contains at least two primers and/or probes thatprovide detectable signals during a polymerase chain reaction, wherein

-   -   the first primer and/or probe provides a detectable signal on        the occurrence on the transcription of host mitochondrial        nucleic acid; and    -   the second primer and/or probe provides a second detectable        signal on the occurrence on the transcription of host nuclear        nucleic acid.

In a third embodiment, a process for identifying a compound or substancethat inhibits viral replication is provided that includes (i) contactingnucleic acid from a virus infected host that has been treated with thecompound with (ii) an amplification reaction mixture that contains atleast two primers and/or probes that provide detectable signals during apolymerase chain reaction, wherein

-   -   the first primer and/or probe provides a detectable signal on        the occurrence of the transcription of viral nucleic acid; and    -   the second primer and/or probe provides a second detectable        signal on the occurrence of the transcription of host nucleic        acid.

In a fourth embodiment, a process for assessing the mitochondrialtoxicity of a compound is provided that includes contacting nucleic acidfrom a host that has been treated with the compound with anamplification reaction mixture that contains at least two primers and/orprobes that provide detectable signals during a polymerase chainreaction, wherein

-   -   the first primer and/or probe provides a detectable signal on        the occurrence on the transcription of host mitochondrial        nucleic acid; and    -   the second primer and/or probe provides a second detectable        signal on the occurrence on the transcription of host nuclear        nucleic acid.

In a fifth embodiment, a process for assessing the tendency of acompound to induce peripheral neuropathy or peripheral lipodystrophy isprovided that includes contacting nucleic acid from a host cell that hasbeen treated with the compound with an amplification reaction mixturethat contains at least two primers and/or probes that provide detectablesignals during a polymerase chain reaction, wherein

-   -   the first primer and/or probe provides a detectable signal on        the occurrence on the transcription of host mitochondrial        nucleic acid; and    -   the second primer and/or probe provides a second detectable        signal on the occurrence on the transcription of host nuclear        nucleic acid.

These processes and methods optimally utilize the conserved regions inthe genome of the virus and host to design unique combinations of a PCRprimer/probe-sets. In one embodiment, this probe contains a detectablesignal, so that upon exonucleic degradation, the signal, indicatingtarget nucleic acid, can be detected in real-time. This technique hasbeen found to be sensitive and accurate; in addition, quantificationusing PCR cycle threshold (Ct) detection during one-step real-timeRT-PCR (Applied Biosystems, CA) has eliminated the variability resultingfrom quantification of end-point RT-PCR products.

In a particular embodiment of the present invention, process ofsimultaneous real-time PCR includes the following steps:

a) contacting at least a portion of a target nucleic acid sequence in asample with

-   -   i) a suitable amplification reaction mixture; and    -   ii) two or more independently labeled oligonucleotides or probes        that hybridizes to the target nucleic acid sequence, such that        the when the target nucleic acid sequence is amplified, each        independently labeled probe releases an unique detectable        signal;    -   iii) wherein at least one independently labeled oligonucleotide        or probe that hydrbiridizes to a target viral nucleic acid        sequence; and    -   iv) at least one independently labeled oligonucleotide or probe        that hydrbiridizes to a target host nucleic acid sequence;        b) carrying out an amplification procedure on the amplification        mixture; and        c) detecting in real time the release of the unique signals.

The presence of the amplicon, of course, indicates that the targetnucleic acid is present in the sample; the target RNA or DNA in thesample can be quantitated based on signal intensity.

The current invention can also be applied to a new method for sensitiveand accurate determination of mitochondrial toxicity of candidatechemotherapeutic compounds using real-time-PCR by determining the ratioof nuclear (or endogenous control) DNA or RNA to mitochondrial DNA orRNA. In a preferred embodiment, this toxicity screening assay is used todetermine toxicity of potential anti-viral agents, and in particularanti-HIV, especially anti-HIV-1, and anti-hepatitis viruses, especiallyHBV and HCV.

In order to quantify the total amount of mitochondrial DNA or RNA,amplification of an endogenous control needs to be performed tostandardize the amount of such mitochondrial DNA or RNA. This endogenouscontrol is an RNA or DNA that is present in each experimental sample andis representative of the total amount of nuclear DNA or RNA. By usingthis endogenous control as an active reference, quantities ofmitochondrial DNA or RNA can be normalized for differences in the amountof total DNA or RNA added to each reaction. Some non-limiting examplesof endogenous controls are any human gene, but especially β-actin,glyceraldehyde-3-phosphate dehydrogenase or ribosomal RNA.

This method includes the following steps:

a) contacting at least a portion of a nuclear nucleic acid sequence in asample with

-   -   i) an amplification reaction mixture; and    -   i) two or more independently labeled oligonucleotides or probes        that hybridizes to the target nucleic acid sequence, such that        the when the target nucleic acid sequence is amplified, each        independently labeled probe releases an unique detectable        signal;    -   ii) wherein at least one independently labeled oligonucleotide        or probe hybridizes to a target nuclear nucleic acid sequence;        and    -   iii) at least one independently labeled oligonucleotide or probe        that hybridizes to a target mitochondrial nucleic acid sequence;        b) carrying out an amplification procedure on the amplification        mixture;        c) detecting in real time the release of the signal.

The quantity of the nuclear amplicon can be compared to the quantity ofmitochondrial amplicon based on differences in signal intensity, therebyindicating the level of mitochondrial toxicity.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is an illustration of a calibration of standard curve for HIV-1(1 a), HCV (1 b), BVDV (1 c), mitochondrial DNA (1 d) and moleculartoxicology (1 e) RT-PCR. The attenuated clinical samples were diluted inDMEM-F12/10% FBS. The Ct value indicates the threshold cycle where theone-step RT-PCR detection of the target becomes positive. The Log cp/mLvalue is the logarithm of the amount of target copies per mL sample. The♦ line indicates the Roche Amplicor HIV-1 Monitor, while the ▪ lineindicates real-time HIV-1 RT-PCR.

FIG. 2 is a graph that depicts the correlation of real-time RT-PCR forHIV-1 with NASBA HIV-1 technology. HIV-1 infected samples were takenfrom SCID-mice experiments. The 99% confidence intervals are indicatedwith dashed lines.

FIG. 3 are illustrations of the effect of antiviral compounds on viralload and RT activity in culture supernatant. The ♦ line indicates datafrom a traditional RT assay, while the ▪ line represents data obtainedfrom using HIV-1 RT-PCR.

FIG. 4 is a non-limiting illustration of RT-PCR standard curves andrelative efficiency plot. In this particular example, quantities ofβ-actin DNA and mitochondrial DNA were measured in real-time to generatethe following plots: 1) the ♦ line is the β-actin standard curve; 2) the▪ line is the mitochondrial DNA standard curve; and 3) the ▴ line is aΔCt plot (Ct β-actin-Ct mitochondrial).

FIG. 5 are illustration of the effect of antiviral compounds onmitochondrial DNA polymerase γ. 2^(−ΔΔCT) is the arithmetic formula usedto express the differences in mitochondrial DNA after calibration (nodrug) and normalization (β-actin). Concentrations are in μM.

FIG. 6 is an illustration of the quantitative detection of viral nucleicacids by real-time PCR. A fluorogenic probe is shown during theextension phase of PCR. If the target sequence is present, the probeanneals downstream from one of the primer sites and is cleaved by the 5′nuclease activity of Taq DNA polymerase as this primer is extended. Thiscleavage of the probe separates the reporter dye from quencher dye,increasing the reporter dye signal. Cleavage removes the probe from thetarget strand, allowing primer extension to continue to the end of thetemplate strand. Thus, inclusion of the probe does not inhibit theoverall PCR process. Additional reporter dye molecules are cleaved fromtheir respective probes with each cycle, effecting an increase influorescence intensity proportional to the amount of amplicon produced.

FIG. 7 is an illustration of the organization of the HCV genome ascompared to the Hepatitis C Virus replicon, indicating the location ofcleavage sites within the polyprotein and the nontranslated regions(NTRs). The open reading frame (ORF) is flanked on the 5′ end by an NTRthat functions as an internal ribosome entry site (IRES) and at the 3′end by a highly conserved sequence essential for genome replication.

FIG. 8 is a graph of the changes in the amounts of cellular and viralnucleic acids over a seven day incubation period in Huh7/HCV Repliconcells.

FIG. 9 is a bar graph of the effect of test compounds on HCV RNA levelsin the Huh7 HCV replicon system.

FIG. 10 contains two bar graphs showing the changes in nucleic acidlevels in Huh7 cells in terms of the amount of mitochondrial RNA and, inthe other, the changes in mitochondrial DNA, after a seven dayincubation period with various drugs.

DETAILED DESCRIPTION OF THE INVENTION

Processes and methods for the simultaneous quantification of nucleicacids in diseased cells that are based on real-time PCR are provided.The real-time-PCR protocol is an excellent tool for reliablequantification of in vitro drug screening and evaluation protocols todetermine the efficacy of potential anti-viral agents. Quantificationusing these simulateous PCR cycle threshold (Ct) detection techniquesduring one-step real-time RT-PCR (Applied Biosysterns, CA) eliminatedthe variability resulting from quantification of end-point RT-PCRproducts. In addition, the mitochondrial toxicity assay is an added toolto assess potential side-effects for these chemotherapeutic agents.

This real time multiplex PCR system includes the simultaneousmeasurements of cellular DNA (for example rDNA) or cellular RNA (forexample rRNA or β-actin m-RNA), and viral RNA or DNA. In one embodiment,the simultaneous real time analysis of host and viral nucleic acidallows the calculation of a sensitivity assay that indicates thecomparative condition of the host cell and the virus. In a separateaspect of the invention, multiplex PCR is used to simultaneously measurethe nuclear and the mitochondrial nucleic acid of a cell to provideinformation on drug toxicity, or to evaluate a cell (in vivo or invitro) that may exhibit a disease that involves mitochondrial toxicity,such as peripheral neuropathy, peripheral lipodystrophy, or a geneticdisease that causes a disruption in mitochondrial DNA or RNA synthesis.

The methods and processes are economic, non-radioactive, rapid,accurate, reproducible, and amenable to large through-put. It canprovide a dynamic range of quantification with linearity of over 5-7logs. One way to express the antiviral effectiveness of a compound is tosubtract the threshold RT-PCR cycle of the test compound with theaverage threshold RT-PCR cycle of the negative control. This value iscalled DeltaCt (ΔCt). A ΔCt of 3.3 equals a 1-log reduction (equalsEC₉₀) in viral nucleic acid production. Compounds that result in areduction of viral nucleic acid greater than 1.5, or more preferred, 2Ct values (75% reduction of viral nucleic acid) are typically usefulcompounds for the inhibition of viral growth.

With the availability of both the viral ΔCt data and the host ΔCt, aspecificity parameter can be introduced. This parameter is obtained bysubtracting the host ΔCt value from the viral ΔCt value. This results inΔΔCt values; a value above 0 means that there is more inhibitory effecton the viral nucleic acid, a ΔΔCt value below 0 means that the hostnucleic acid is more affected. As a general rule, ΔΔCt values above 2are considered as significantly different from the no-drug treatmentcontrol, and hence, exhibits useful antiviral activity. However,compounds with a ΔΔCt value of less than 2, but showing limitedmolecular cytotoxicty data (rRNA ΔCT between 0 and 2) may also bedesired for certain applications requiring compounds with low toxicity.

As an example, a compound might reduce the host RNA polymerase activity,but not the host DNA polymerase activity. Therefore, quantification ofrDNA or β-actin DNA (or any other host DNA fragment) and comparison withDNA levels of the no-drug control is a relative measurement of theinhibitory effect of the test compound on cellular DNA polymerases. Withthe availability of both the HCV ΔCt data and the rDNA ΔCt, aspecificity parameter can be introduced. This parameter is obtained bysubtracting both ΔCt values from each other. This results in ΔΔCtvalues; a value above 0 means that there is more inhibitory effect onthe viral encoded polymerase, a ΔΔCt value below 0 means that the hostrDNA levels are more affected than the viral nucleic acid levels. As ageneral rule, ΔΔCt values above 2 are considered as significantlydifferent from the no-drug treatment control, and hence, is aninterested compound for further evaluation. However, compounds with aΔΔCt value of less than 2, but with limited molecular cytotoxicty (rDNAΔCT between 0 and 2) are also possible active candidate compounds forfurther evaluation

In a first embodiment, a process for assessing a viral disease isprovided that includes contacting nucleic acid from a viral infectedhost cell with an amplification reaction mixture that contains at leasttwo primers and/or probes that provide detectable signals during apolymerase chain reaction, wherein

-   -   the first primer and/or probe provides a detectable signal on        the occurrence on the transcription of host nucleic acid; and    -   the second primer and/or probe provides a second detectable        signal on the occurrence on the transcription of viral nucleic        acid.

In a particular embodiment, the level of transcription of the viral andhost nucleic acid is compared to that of a standard, including but notlimited to, a known viral infected host cell, or alternatively, aninternal standard can be established by comparing the extent oftranscription of the host and viral nucleic acid over a number ofsamples from the host to monitor and measure the change in infection. Inanother embodiment, the data can be assessed as described above throughthe use of ΔCT and ΔΔCt values.

In a preferred embodiment, the nucleic acid is a consensus or non-codingsequence, which can be either 5′ or 3′ to the target expressed sequence.In one embodiment, the non-coding sequence is an intron or a partthereof. Non-limiting examples are non-coding sequences from β-actin orGAPDH.

The host nucleic acid can be nuclear or cytoplasmic, and in particular,mitochondrial nucleic acid, and the viral nucleic acid can be either DNAor RNA.

This process can be used to evaluate the ability of the compound orsubstance to inhibit the replication of any virus, including but notlimited to a virus from the Retroviridae, Flaviviridae,Orthomyxoviridae, Paramyxoviridae, Herpesviridae, Hepadnaviridae,Picornaviridae, Reoviridae, Poxyiridae, Adenoviridae, Papoviridae,Parvoviridae, Bunyaviridae, Filoviridae, Arenaviridae or Togaviridaefamily. In particular, the virus is HIV, hepatitis (including but notlimited to A, B, C, D and G), BVDV (bovine diarrhea virus), herpessimplex, Adenovirus type 1, influenza, including influenza A (HINI),influenza A (H3N2), influenza B, influenza C and influenza D, measles,mumps, parainfluenza type 3, RSV (respiratory syncytial virus), HSV(herpes simplex virus), EBV (Epstein Barr virus), CMV (cytomegalovirus)or West Nile Virus.

In a second embodiment, a process for assessing a disease state thatincludes a disruption in mitochondrial DNA or RNA synthesis is providedthat includes contacting nucleic acid from a host with an amplificationreaction mixture that contains at least two primers and/or probes thatprovide detectable signals during a polymerase chain reaction, wherein

-   -   the first primer and/or probe provides a detectable signal on        the occurrence on the transcription of host mitochondrial        nucleic acid; and    -   the second primer and/or probe provides a second detectable        signal on the occurrence on the transcription of host nuclear        nucleic acid.

In a third embodiment, a process for identifying a compound or substancethat inhibits viral replication is provided that includes (i) contactingnucleic acid from a virus infected host that has been treated with thecompound with (ii) an amplification reaction mixture that contains atleast two primers and/or probes that provide detectable signals during apolymerase chain reaction, wherein

-   -   the first primer and/or probe provides a detectable signal on        the occurrence of the transcription of viral nucleic acid; and    -   the second primer and/or probe provides a second detectable        signal on the occurrence of the transcription of host nucleic        acid.

In a fourth embodiment, a process for assessing the mitochondrialtoxicity of a compound is provided that includes contacting nucleic acidfrom a host that has been treated with the compound with anamplification reaction mixture that contains at least two primers and/orprobes that provide detectable signals during a polymerase chainreaction, wherein

-   -   the first primer and/or probe provides a detectable signal on        the occurrence on the transcription of host mitochondrial        nucleic acid; and    -   the second primer and/or probe provides a second detectable        signal on the occurrence on the transcription of host nuclear        nucleic acid.

In a fifth embodiment, a process for assessing the tendency of acompound to induce peripheral neuropathy or peripheral lipodystrophy isprovided that includes contacting nucleic acid from a host cell that hasbeen treated with the compound with an amplification reaction mixturethat contains at least two primers and/or probes that provide detectablesignals during a polymerase chain reaction, wherein

-   -   the first primer and/or probe provides a detectable signal on        the occurrence on the transcription of host mitochondrial        nucleic acid; and    -   the second primer and/or probe provides a second detectable        signal on the occurrence on the transcription of host nuclear        nucleic acid.

These processes and methods optimally utilize the conserved regions inthe genome of the virus and host to design unique combinations of a PCRprimer/probe-sets. In one embodiment, this probe contains a detectablesignal, so that upon exonucleic degradation, the signal, indicatingtarget nucleic acid, can be detected in real-time. This technique hasbeen found to be sensitive and accurate; in addition, quantificationusing PCR cycle threshold (Ct) detection during one-step real-timeRT-PCR (Applied Biosystems, CA) has eliminated the variability resultingfrom quantification of end-point RT-PCR products.

In a particular embodiment of the present invention, a method ofsimultaneous real-time PCR includes the following steps:

a) contacting at least a portion of a target nucleic acid sequence in asample comprising:

-   -   i) a suitable amplification reaction mixture; and    -   ii) two or more independently labeled oligonucleotides or probes        that hybridizes to the target nucleic acid sequence, such that        the when the target nucleic acid sequence is amplified, each        independently labeled probe releases an unique detectable        signal;    -   iii) wherein at least one independently labeled oligonucleotide        or probe that hydrbiridizes to a target viral nucleic acid        sequence; and    -   iv) at least one independently labeled oligonucleotide or probe        that hydrbiridizes to a target host nucleic acid sequence;        b) carrying out an amplification procedure on the amplification        mixture; and        c) detecting in real time the release of the unique signals.

The presence of the amplicon, of course, indicates that the targetnucleic acid is present in the sample; the target RNA or DNA in thesample can be quantitated based on signal intensity.

The current invention can also be applied to a new method for sensitiveand accurate determination of mitochondrial toxicity of candidatechemotherapeutic compounds using real-time-PCR by determining the ratioof nuclear (or endogenous control) DNA or RNA to mitochondrial DNA orRNA. In a preferred embodiment, this toxicity screening assay is used todetermine toxicity of potential anti-viral agents, and in particularanti-HIV, especially anti-HIV-1, and anti-hepatitis viruses, especiallyHBV and HCV.

This method includes the following steps:

a) contacting at least a portion of a nuclear nucleic acid sequence in asample comprising:

-   -   i) an amplification reaction mixture; and    -   i) two or more independently labeled oligonucleotides or probes        that hybridizes to the target nucleic acid sequence, such that        the when the target nucleic acid sequence is amplified, each        independently labeled probe releases an unique detectable        signal;    -   ii) wherein at least one independently labeled oligonucleotide        or probe hybridizes to a target nuclear nucleic acid sequence;        and    -   iii) at least one independently labeled oligonucleotide or probe        that hybridizes to a target mitochondrial nucleic acid sequence;        d) carrying out an amplification procedure on the amplification        mixture;        e) detecting in real time the release of the signal.

The quantity of the nuclear amplicon can be compared to the quantity ofmitochondrial amplicon based on differences in signal intensity, therebyindicating the level of mitochondrial toxicity.

I. Screening

These processes and methods can be used to evaluate the ability of thecompound or substance to inhibit the replication of any virus, includingbut not limited to a virus from the Retroviridae, Flaviviridae,Orthomyxoviridae, Paramyxoviridae, Herpesviridae, Hepadnaviridae,Picornaviridae, Reoviridae, Poxyiridae, Adenoviridae, Papoviridae,Parvoviridae, Bunyaviridae, Filoviridae, Arenaviridae or Togaviridaefamily. In particular, the virus is HIV, hepatitis (including but notlimited to A, B, C, D and G), BVDV (bovine diarrhea virus), herpessimplex, Adenovirus type 1, influenza, including influenza A (HINI),influenza A (H3N2), influenza B, influenza C and influenza D, measles,mumps, parainfluenza type 3, RSV (respiratory syncytial virus), HSV(herpes simplex virus), EBV (Epstein Barr virus), CMV (cytomegalovirus)or West Nile Virus.

In particular, quantitative real-time PCR antiviral screening can becombined with calibration for a host RNA targets (in RT-PCR) in thefollowing non-limiting examples:

-   a) anti-HCV compound screening can be combined with rRNA    calibration, mRNA calibration, and in particular -actin mRNA    calibration, mitochondrial RNA calibration and/or any other nuclear    or mitochondrial nucleic acid calibration;-   b) anti-HIV compound screening can be combined with rRNA    calibration, mRNA calibration, and in particular -actin mRNA    calibration, mitochondrial RNA calibration and/or any other nuclear    or mitochondrial nucleic acid calibration;-   c) anti-HBV compound screening can be combined with rRNA    calibration, mRNA calibration, and in particular -actin mRNA    calibration, mitochondrial RNA calibration and/or any other nuclear    or mitochondrial nucleic acid calibration;-   d) anti-RSV compound screening can be combined with rRNA    calibration, mRNA calibration, and in particular -actin mRNA    calibration, mitochondrial RNA calibration and/or any other nuclear    or mitochondrial nucleic acid calibration;-   e) anti-BVDV compound screening can be combined with rRNA    calibration, mRNA calibration, and in particular -actin mRNA    calibration, mitochondrial RNA calibration and/or any other nuclear    or mitochondrial nucleic acid calibration;-   f) anti-lentivirus compound screening can be combined with rRNA    calibration, mRNA calibration, and in particular -actin mRNA    calibration, mitochondrial RNA calibration and/or any other nuclear    or mitochondrial nucleic acid calibration;-   g) anti-flaviviridae (Flavivirus, Hepacivirus, Pestivirus) compound    screening can be combined with rRNA calibration, mRNA calibration,    and in particular -actin mRNA calibration, mitochondrial RNA    calibration and/or any other nuclear or mitochondrial nucleic acid    calibration;-   h) anti-hepadnavirus compound screening can be combined with rRNA    calibration, mRNA calibration, and in particular -actin mRNA    calibration, mitochondrial RNA calibration and/or any other nuclear    or mitochondrial nucleic acid calibration;-   i) anti-picornavirus compound screening can be combined with rRNA    calibration, mRNA calibration, and in particular -actin mRNA    calibration, mitochondrial RNA calibration and/or any other nuclear    or mitochondrial nucleic acid calibration;-   j) anti-herpetoviridae (HSV, HCMV, EBV) compound screening can be    combined with rRNA calibration, mRNA calibration, and in particular    -actin mRNA calibration, mitochondrial RNA calibration and/or any    other nuclear or mitochondrial nucleic acid calibration.

Quantitative real-time PCR antiviral screening can be combined withcalibration for a host DNA target (in PCR) in the following non-limitingexamples:

-   a) anti-HCV compound screening can be combined with rDNA    calibration, DNA calibration, and in particular -actin DNA    calibration, mitochondrial DNA calibration and/or any other nuclear    or mitochondrial nucleic acid calibration;-   b) anti-HIV compound screening can be combined with rDNA    calibration, DNA calibration, and in particular -actin DNA    calibration, mitochondrial DNA calibration and/or any other nuclear    or mitochondrial nucleic acid calibration;-   c) anti-HBV compound screening can be combined with rDNA    calibration, DNA calibration, and in particular -actin DNA    calibration, mitochondrial DNA calibration and/or any other nuclear    or mitochondrial nucleic acid calibration;-   d) anti-RSV compound screening can be combined with rDNA    calibration, DNA calibration, and in particular -actin DNA    calibration, mitochondrial DNA calibration and/or any other nuclear    or mitochondrial nucleic acid calibration;-   e) anti-BVDV compound screening can be combined with rDNA    calibration, DNA calibration, and in particular -actin DNA    calibration, mitochondrial DNA calibration and/or any other nuclear    or mitochondrial nucleic acid calibration;-   f) anti-lentivirus compound screening can be combined with rDNA    calibration, DNA calibration, and in particular -actin DNA    calibration, mitochondrial DNA calibration and/or any other nuclear    or mitochondrial nucleic acid calibration;-   g) anti-flaviviridae (Flavivirus, Hepacivirus, Pestivirus) compound    screening can be combined with rDNA calibration, DNA calibration,    and in particular -actin DNA calibration, mitochondrial DNA    calibration and/or any other nuclear or mitochondrial nucleic acid    calibration;-   h) anti-hepadnavirus compound screening can be combined with rDNA    calibration, DNA calibration, and in particular -actin DNA    calibration, mitochondrial DNA calibration and/or any other nuclear    or mitochondrial nucleic acid calibration;-   i) anti-picomavirus compound screening can be combined with rDNA    calibration, DNA calibration, and in particular -actin DNA    calibration, mitochondrial DNA calibration and/or any other nuclear    or mitochondrial nucleic acid calibration;-   j) anti-herpetoviridae (HSV, HCMV, EBV) compound screening can be    combined with rDNA calibration, DNA calibration, and in particular    -actin DNA calibration, mitochondrial DNA calibration and/or any    other nuclear or mitochondrial nucleic acid calibration.

The current invention also provides a new process and method forsensitive and accurate determination of mitochondrial toxicity ofchemotherapeutic or other pharmaceutical agents by determining the ratioof mitochondrial DNA or RNA to nuclear DNA or RNA. The rationale behindthis methodology is driven by the fact that DNA polymerase γ inhibitioneventual leads to lower amounts of mitochondrial DNA or RNA, while theamounts of nuclear DNA or RNA (for which replication is dependent on DNApolymerase α and/or β) remains constant.

In order to quantify the total amount of mitochondrial DNA or RNA,amplification of an endogenous control needs to be performed tostandardize the amount of such mitochondrial DNA or RNA. This endogenouscontrol is an RNA or DNA that is present in each experimental sample andis representative of the total amount of nuclear DNA or RNA. By usingthis endogenous control as an active reference, quantities ofmitochondrial DNA or RNA can be normalized for differences in the amountof total DNA or RNA added to each reaction. Endogenous controls can beany human gene, but often β-actin, glyceraldehyde-3-phosphatedehydrogenase, or ribosomal RNA have been used. An effective process toquantify the total amount of endogenous control in a reaction byreal-time PCR is provided

II. Definitions

As used herein, “sample” or “clinical sample” relates to any sampleobtained from a host for use in carrying out the procedures of thepresent invention. In one aspect, the host is suffering from a diseaseor syndrome that is at least partially caused by a virus. The host mayalso be an asymptomatic considered to be at risk of viral infection. Thesample may be a cellular sample such as a tissue sample, for example oflung tissue obtained as a biopsy or post-mortem, a fluid sample, such asblood, saliva, sputum, urine, cerebrospinal fluid, or a swabbed sampleobtained by swabbing a mucus membrane surface such as nasal surface, apharyngeal surface, a buccal surface, and the like, or it may beobtained from an excretion such as feces, or it may be obtained fromother bodily tissues or body fluids commonly used in diagnostic testing.

The term “purified” in reference to RNA or DNA, as used herein, relatesto released RNA or DNA from latent or inaccessible form in a virion or acell and allowing the RNA or DNA to become freely available. In such astate, it is suitable for effective amplification by use of thepolymerase chain reaction. Releasing RNA or DNA may include steps thatachieve the disruption of virions containing viral RNA or DNA, as wellas disruption of cells that may harbor such virions. Purification of RNAor DNA is generally carried out under conditions that rigorously andeffectively exclude or inhibit any nuclease activity that may bepresent. Additionally, purification may include steps that achieve atleast a partial separation of the RNA or DNA dissolved in an aqueousmedium from other cellular or viral components, wherein such componentsmay be either particulate or dissolved.

As used herein, “reverse transcription” or “RT” relates to a procedurecatalyzed by an enzyme, reverse transcriptase, that synthesizes a cDNAfrom a single stranded RNA molecule, with the use of oligonucleotideprimers having free 3′-hydroxyl groups. As used herein, the term“polymerase chain reaction” or “PCR” relates to a procedure whereby alimited segment of a nucleic acid molecule, which frequently is adesired or targeted segment, is amplified repetitively to produce alarge amount of DNA molecules which consist only of that segment. Theprocedure depends on repetition of a large number of priming andtranscription cycles. In each cycle, two oligonucleotide primers bind tothe segment, and define the limits of the segment. A primer-dependantDNA polymerase then transcribes, or replicates, the strands to which theprimers have bound. Thus, in each cycle, the number of DNA duplexes isdoubled.

The term “primer” or “oligonucleotide primer,” as used herein, relatesto an oligonucleotide having a specific or desired nucleotide sequencethat is complementary to a particular sequence on one of the strands ofa DNA duplex. When the primer is caused to hybridize to the specificsequence in a DNA duplex to which it is complimentary, it may serve asthe priming position, or the initiation position, for the action of aprimer-dependent DNA polymerase activity. The primer, once hybridized,acts to define the 5′-end of the operation of the transcription activityof the polymerase on the duplex. Commonly in PCR, a specific pair ofprimers is employed, wherein one of the primers hybridizes to one of thestrands and the second primer hybridizes to the complementary strand.The primers hybridize in such an orientation that transcription, whichproceeds in the direction from 5′ to 3′, is in the direction leadingfrom each primer toward the site of hybridization of the other primer.After several rounds of hybridization and transcription the amplifiedDNA produced is a segment having a defined length whose ends are definedby the sites to which the primers hybridize.

The term “probe” or “labeled oligonucleotide,” as used herein, relatesto an oligonucleotide having a specific or desired nucleotide sequencethat is complementary to a particular sequence on one of the strands ofa DNA duplex, as well as a detectable signal, such as a fluorescent dye.When the primer is caused to hybridize to the specific sequence in a DNAduplex to which it is complimentary, the signal is inactive, for exampledue to a covalently linked quenching dye. However, upon amplificationand subsequent analysis, the signal is activated by exonucleicdegradation and thus can be detected in real time. In particular, theprobe can contain a fluorescent dye and a quenching dye, such that atthe time of hybridization, the fluorescent dye in quenched by thequenching dye. After amplification and exonucleic degradation, thefluorescent dye is released from the quenching dye and a fluorescentsignal can be detected in real time.

The term “amplification reaction mixture,” as used herein refers to anyreaction substance, or combination of substances that promotes theamplification of a target nucleic acid sequence, including enzymes suchas polymerase, or polymerases with exonuclease activity, substrates suchas nucleic acids and oligonucleotide primers, as defined herein.

As used herein, the term “specific to” or “specific for” a targetsequence, in relation to a nucleic acid sequence such as anoligonucleotide sequence, relate to a nucleotide sequence thathybridizes, under conditions used in given experimental circumstances,to the target but does not hybridize under those circumstances tosequences that are not target sequences. Nucleotide sequences that arespecific for a particular target, such as the HIV target sequences thatare included in the subject matter of the present invention, are thosethat include bases all of which are complementary to the correspondingbase on the target.

Further, the term “specificity,” as used herein, of a nucleic acidsequence for a target sequence also encompasses nucleic acids andoligonucleotides having a small number of nucleotides that may not becomplementary to the corresponding nucleotides of the target sequence.Such sequences are still “specific” for the target sequence, as long asthe extent of the deviation from complementarity remains functionally ofno consequence. In particular, such a sequence is “specific” for thetarget sequence as long as it hybridized effectively to the targetsequence but does not hybridize to any sequence that is not a targetsequence in the sample, under the conditions used in given experimentalcircumstances.

The term “amplicon” as used herein refers to a double stranded nucleicacid segment having a defined size and sequence that results from anamplification procedure, such as a PCR procedure. The size of theamplicon is limited by the sites on the two strands of a nucleic acidduplex to which the primers bind. That segment of the product nucleicacid becomes the prevalent product of the amplification procedure aftera small number of cycles of amplification.

The term “host,” as used herein, refers to a unicellular ormulticellular organism in which the virus can replicate, including celllines and animals, and preferably a human. Alternatively, the host canbe carrying a part of the viral genome, whose replication or functioncan be altered by the compounds of the present invention. The term hostspecifically refers to infected cells, cells transfected with all orpart of the viral genome and animals, in particular, primates (includingchimpanzees) and humans. In most animal applications of the presentinvention, the host is a human patient. Veterinary applications, incertain indications, however, are clearly anticipated by the presentinvention (such as bovine viral diarrhea virus in cattle, hog choleravirus in pigs, and border disease virus in sheep).

III. Host Primers and Probes

For the detection of host nucleic acids, any suitable primer and/orprobe known in the art may be used. These primers and/or probes may bepurchase or made by any means known in the art. There are severalprimers and/or probe combinations commercially available, for examplethe primer probe set for rRNA gene (Perkin Elmer/Applied Biosystems).The latter set is often used as calibrator PCR in this invention.Alternatively, suitable probes and primers can be designed by using thePrimer Express software (Applied Biosysterns, CA), and in particular newprimers and probes for the β-actin gene, and for the mitochondrialcytochrome oxidase subunit II (COXII) gene.

β-Actin

In one embodiment, the nuclear DNA or RNA used to derive a set ofoligonucleotides for the endogenous control is the DNA for β-actin. Anysuitable primers and/or probes can be used. In a specific embodiment ofthe present invention, the primers and/or probes are complementary tosequences from the third exon of the human -actin gene (GenBandkaccession number E01094). The probe comprises a reporter and quencherthat provides a detectable signal upon amplification. Anyreporter/quencher probe set can be used, including, but not limited toTaqMan, molecular beacons, single dye probe, SYBR green, Amplifluorprobes and dual labeled probe sets.

In a preferred embodiment of the invention, the oligonucleotides used toamplify β-actin (primers) are sense sense 5′-GCGCGGCTACAGCTTCA-3′(Sequence ID No. 1) and antisense 5′-TCTCCTTAATGTCACGCACGAT-3′ (SequenceID No. 2). The labeled oligonulceotide (probe) used to detect hostnucleic acid has a sequence of 5′-CACCACGGCCGAGCGGGA-3′ (Sequence ID No.3). In one embodiment, the probe is labeled with a reporter at the5′-end and a quencher molecule at the 3′-end, and in particular, thereporter, FAM, at the 5′ end, and the quencher molecule, TAMRA, at the3′ end.

Mitochondiral Nucleic Acid

In one embodiment, the mitochondrial nucleic acids can be specificallyderived from mitochondrial DNA. In an alternate embodiment, themitochondiral nucleic acids can be specifically derived frommitochondrial RNA. In an alternate embodiment, the mitochondiral nucleicacids are complementary to sequences from themitochondrial COXII gene.Any suitable primers and/or probes can be used. The probe comprises areporter and quencher that provides a detectable signal uponamplification. Any reporter/quencher probe set can be used, including,but not limited to TaqMan, molecular beacons, single dye probe, SYBRgreen, Amplifluor probes and dual labeled probe sets.

In a preferred embodiment of the invention, the oligonucleotides used toamplify mitochondrial nucleic acids (primers) are sense sense sense5′-TGCCCGCCATCATCCTA-3′ (Sequence ID No. 19) and5′-TCGTCTGTTATGTAAAGGATGCGT-3′ (Sequence ID No. 20). The labeledoligonulceotide (probe) used to detect host nucleic acid has a sequenceof 5′-TCCTCATCGCCCTCCCATCCC-3′ (Sequence ID No. 21). In one embodiment,the probe is labeled with a reporter at the 5′-end and a quenchermolecule at the 3′-end, and in particular, the reporter, TET, at the 5′end, and the quencher molecule, TAMRA, at the 3′ end.

IV. Viral Primers and Probes

For viral targets, any suitable primer and/or probe known in the art maybe used. These primers and/or probes may be purchase or made by anymeans known in the art. Alternatively, suitable probes and primers canbe designed by using the Primer Express software (Applied Biosystems,CA), and in particular, primers and probes designed to be complementaryto highly conserved areas. This is particularly important for viruseswith a high genetic variability, like for example HCV, HBV, and HIV,BVDV and RSV.

Ideally, the viral primer/probe set should fulfill to the followingcriteria: (i) be able to detect the huge variability of clades orgenotypes with the same efficiency; ii) have a dynamic range of at leastfive logs or higher; and iii) the lower limit of detection should be aslow as a few viral copies/mL. Although variability at the PCR-primerbinding sites is often problematic, RT-PCR based assays are some of themost sensitive technologies.

In one embodiment of the present invention, complementary viralsequences were designed based on conserved regions of the viral genometo obtain a unique combination of PCR primers and/or probe-set. In analternative embodiment of the present invention, the primers/probes aredesigned based on predicted sequence conservation over the differentgenotypes. In a preferred embodiment, the primers/probes are designedbased on both the conserved region of the viral genome and predictedsequence conservation over the different genotypes.

HIV

In one embodiment of the invention, the target viral nucleic acid isfrom HIV, and in particular, HIV-1. Any suitable primers and/or probescan be used. In a specific embodiment of the present invention, theprimers and/or probes are complementary to the reverse transcriptasedomain between codons 200 and 280. The probe comprises a reporter andquencher that provides a detectable signal upon amplification. Anyreporter/quencher probe set can be used, including, but not limited toTaqMan, molecular beacons, single dye probe, SYBR green, Amplifluorprobes and dual labeled probe sets.

In a preferred embodiment of the invention, the oligonucleotides used toamplify HIV-1 (primers) are sense 5′-TGGGTTATGAACTCCATCCTGAT-3′(Sequence ID No. 4) and antisense 5′-TGTCATTGACAGTCCAGCTGTCT-3′(Sequence ID No. 5). The labeled oligonulceotide (probe) used to detectHIV-1 viral load has a sequence of 5′-TTTCTGGCAGCTCTCGGCTGTACTGTCCATT-3′(Sequence ID No. 6). In one embodiment, the probe is labeled with areporter at the 5′-end and a quencher molecule at the 3′-end, and inparticular, the reporter, FAM, at the 5′ end, and the quencher molecule,TAMRA, at the 3′ end.

HCV

In another embodiment of the invention, the target viral nucleic acid isfrom HCV. Any suitable primers and/or probes can be used. In a specificembodiment of the present invention, the primers and/or probes arederived from thighly conserved sequences complementary to the RNAsequences present in HCV, such as the HCV 5′ non-coding region. Theprobe comprises a reporter and quencher that provides a detectablesignal upon amplification. Any reporter/quencher probe set can be used,including, but not limited to TaqMan, molecular beacons, single dyeprobe, SYBR green, Amplifluor probes and dual labeled probe sets.

In a preferred embodiment of the invention, the oligonucleotides used toamplify HCV (primers) are sense 5′-AGCCATGGCGTTAGTA(T/A)GAGTGT-3′(Sequence D No. 7) and antisense 5′-TTCCGCAGACCACTATGG-3′ (Sequence IDNo. 8). The labeled oligonulceotide (probe) used to detect HCV viralload has a sequence of 5′-CCTCCAGGACCCCCCCTCCC-3′ (Sequence ID No. 9).In one embodiment, the probe is labeled with a reporter at the 5′-endand a quencher molecule at the 3′-end, and in particular, the reporter,FAM, at the 5′ end, and the quencher molecule, TAMRA, at the 3′ end.

BVDV

In another embodiment of the invention, the target viral nucleic acid isfrom BVDV. Any suitable primers and/or probes can be used. In a specificembodiment of the present invention, the primers and/or probes arederived from thighly conserved sequences complementary, such assequences complementary to nucleotides 1611 to 1751 of the NS5B gene.The probe comprises a reporter and quencher that provides a detectablesignal upon amplification. Any reporter/quencher probe set can be used,including, but not limited to TaqMan, molecular beacons, single dyeprobe, SYBR green, Amplifluor probes and dual labeled probe sets.

In a preferred embodiment of the invention, the oligonucleotides used toamplify BVDV (primers) are sense sense 5′-AGTCTTCAGTTTCTTGCTGATGT-3′(Sequence ID No. 10) and antisense 5′-TGTTGCGAAAGGACCAACAG-3′ (SequenceID No. 11). The labeled oligonulceotide (probe) used to detect BVDVviral load has a sequence of 5′-AAATCCTCCTAACAAGCGGGTTCCAGG-3′ (SequenceID No. 12). In one embodiment, the probe is labeled with a reporter atthe 5′-end and a quencher molecule at the 3′-end, and in particular, thereporter, FAM, at the 5′ end, and the quencher molecule, TAMRA, at the3′ end.

HBV

In another embodiment of the invention, the target viral nucleic acid isfrom HBV. Any suitable primers and/or probes can be used. In a specificembodiment of the present invention, the primers and/or probes arederived from thighly conserved sequences complementary to the DNAsequences present in HBV, such as the amino-terminal region of the HBVsurface antigen gene. The probe comprises a reporter and quencher thatprovides a detectable signal upon amplification. Any reporter/quencherprobe set can be used, including, but not limited to TaqMan, molecularbeacons, single dye probe, SYBR green, Amplifluor probes and duallabeled probe sets.

In a preferred embodiment of the invention, the oligonucleotides used toamplify HBV (primers) are sense sense 5′-GGACCCCTGCTCGTGTTACA-3′(Sequence ID. No. 13) and antisense 5′-GAGAGAAGTCCACCACGAGTCTAG-3′(Sequence ID No. 14). The labeled oligonulceotide (probe) used to detectHBV viral load has a sequence of5′-TGTTGACAA(A/G)TCCTCACAATACC(A/G)CAGA-3′ (Sequence ID No. 15). In oneembodiment, the probe is labeled with a reporter at the 5′-end and aquencher molecule at the 3′-end, and in particular, the reporter, FAM,at the 5′ end, and the quencher molecule, TAMRA, at the 3′ end.

RSV

In another embodiment of the invention, the target viral nucleic acid isfrom RSV. Any suitable primers and/or probes can be used. In a specificembodiment of the present invention, the primers and/or probes arederived from thighly conserved sequences complementary, such assequences complementary to nucleotides that encode for the RNApolymerase large subunit (L). The probe comprises a reporter andquencher that provides a detectable signal upon amplification. Anyreporter/quencher probe set can be used, including, but not limited toTaqMan, molecular beacons, single dye probe, SYBR green, Amplifluorprobes and dual labeled probe sets.

In a preferred embodiment of the invention, the oligonucleotides used toamplify RSV (primers) are sense sense sense5′-CAACAACCCTAATCATGTGGTATCA-3′ (Sequence ID No. 16) and antisense57-CCGGTTGCATTGCAAACA-3′ (Sequence ID No. 17). The labeledoligonulceotide (probe) used to detect RSV viral load has a sequence of5′-TGACAGGCAAAGAAAGAGAACTCAGTGTAGGTAGA-3′ (Sequence ID No. 18). In oneembodiment, the probe is labeled with a reporter at the 5′-end and aquencher molecule at the 3′-end, and in particular, the reporter, FAM,at the 5′ end, and the quencher molecule, TAMRA, at the 3′ end.

V. Methods

Amplification Procedure

The process for amplification of a desired nucleic acid sequence can beachieve by any means necessary to achieve amplification of the desiredamplicon. The amplification can be achieved using any known means in theart, including polymerase chain reaction techniques. The primers andprobes can be purchased or prepared by any means known in the art,including automated processes. In a preferred embodiment, the primersand probes are designed for specificity for the target nucleic acidsequence, as disclosed herein. The enzymes used to promote amplificationcan be purchased or can be prepared by any means known in the art,including cellular extraction. Substrates to aid in the amplificationcan also be purchased or can be prepared by any means known in the art,including any synthetic methodology to synthesis natural and unnaturalnucleic acids. The enzyme and substrates can be added to theamplification mixture at any time and order that allows for theamplification of the desired amplicon. In a preferred embodiment, thepolymerase and substrates follow TaqMan 7700 chemistry provided byApplied Biosystems in California.

Additionally, amplification conditions vary depending on the choice ofprimers and probes, due to differences in their melting temperatures™.Preferred temperatures are from 50° C. to 95° C. for incubation and 60°C. to 95° C. for amplification. The temperature for amplification can bedone at any temperature that allows for replication of the desiredamplicon at a suitable rate. As an exemplary embodiment,reverse-transcriptase polymerase chain reaction (“RT-PCT”) can be usedto amplify the desired amplicon. After reverse transcription incubation,an amplification cycle can be performed. The incubation cycle can beperformed at one temperature or on a multi-temperature basis; forexample, the incubation cycle can be performed on a two-step temperaturegradient, preferably, first a moderate time at moderate temperaturefollowed by an extended period at higher temperatures. The amplificationcycle can be performed at one temperature or on a multi-temperaturebasis; for example, the amplification cycle can be performed on atwo-step gradient, preferably, first a short phase of highertemperatures followed by a longer period of moderate temperatures. Theamplification procedure can be repeated as many times as necessary, butpreferably repeated around 40 times.

As a non-limiting example, HIV-1, β-actin and mitochondrial nucleic acidsequences can be amplified using the following procedure. First theamplification reaction mixture is incubated for two minutes at 50° C.,then ten minutes at 95° C. This is then followed by forty cycles of atwo-step amplification reaction at 95° C. for fifteen seconds then sixtyseconds at 60° C.

Detection Systems

The presence of the amplicon can be detected in real time based on thelabeled oligonucleotide, which is labeled with a variety of substances,termed reporting dyes, and quenching dye, which upon amplification, arecapable of emitting a detectable signal. Any combination of reportingdyes and quenching dyes can be used. Some non-limiting examples ofreporting dyes are FAM, VIC, PAT and JOE. A non-limiting example ofquenching dyes is TAMRA. These reporting dyes and quenching dyes can bepurchased or can be prepared by any means known in the art, includingradical and organometallic chemistry.

In one embodiment, the detectable signal is a fluorescent dye that canbe detected in a spectrometer that is covalently bound to a quenchingdye through the oligonucleotide. This renders the fluorescent dyeinactive while bound to the oligonucleotide. However, upon exonucleasedegradation of the oligonucleotide, the fluorescent dye can be releasedfrom the quenching dye, thus emitting a detectable signal.

Many of the new DNA tags and labels depend on two phenomena that areextensions of fluorescence: quenching and energy transfer. In general,anything that reduces the lifetime of the excited state decreases thequantum yield of the fluorophore; anything that decreases the quantumyield is called quenching. There are three main mechanisms fordetermining these phenomena: collisional, in which the excited state ofthe fluorophore loses its energy by bumping into a nonfluorescentmolecule; static, in which the excited state reacts with the quencher,forming a nonfluorescent complex; and energy transfer, which involvesthe nonradiative transfer of energy from a donor to an acceptor.

The brightness of a fluorescent dye depends on many parameters. Theparameters can be divided between the physical and chemical propertiesof the dyes and the excitation system. The important physical propertiesof the dyes are quantum yield and extinction coefficient. The quantumyield is an expression of the number of photons emitted divided by thenumber of photons absorbed. A quantum yield of 0 indicates anonfluorescent molecule, and a quantum yield of 1 indicates that 100percent of the excitation photons result in lower-wavelength emittedphotons. The extinction coefficient is an expression of the probabilitythat a photon of a given wavelength will be absorbed by the fluorophore.A high extinction coefficient combined with a high quantum yieldgenerally leads to a “bright” fluorophore; fluorescein, for example, isa relatively “bright” dye, having an extinction coefficient of about80,000 at its absorption maximum and a quantum yield of ˜0.9.

For fluorescence resonant energy transfer (FRET) to occur, there must bea precise overlap in quantum energy levels between the donor and theacceptor, the energy being transferred by dipolar coupling rather thanemission and reabsorption of a photon. FRET has been used veryproductively to create dyes for DNA sequencing, where a common donoreliminates the need for multiple excitation wavelengths but insteadtransfers its energy to four separate dyes that have easily discernableemission spectra. FRET and fluorescence quenching are very distancedependant, allowing their exploitation in several novel assays thatalter donor-acceptor geometries.

Many of the methods described depend on a variety of modifiedoligonucleotides. Many fluorescent dyes are available asdye-phosphoramidites (or as dye-CPG derivatives), which are compatiblewith automated oligonucleotide synthesis methods. Using this approach,dyes can be incorporated at the 5′ or 3′ end or at any internal positionduring routine synthesis. Similarly, amino-modified bases can beincorporated into an oligo at any position, enabling a wider variety oflabeling, because many additional dyes are available in an NHS-esterform that can be conjugated to an amino-modified oligonucleotide aftersynthesis. Different applications call for different modifications,including such esoterica as variable-length spacers, universal bases andbranched backbones.

Reagent kits that support quantitative amplification and detection inmultiplex are commercially available. The QPCR kits are used with DNAtemplates, either to detect DNA mutations or to measure gene or viralcopy number. The QRT-PCR kits are used with RNA templates, typically formeasuring RNA levels. Mutations can also be detected in expressed RNAwith these kits. These kits have the capability of high performance withvarious fluorescent detection systems, including, the AmpliFluor system,molecular beacons, TaqMan® probes, dual fluorophore approach, single-dyeprimers and DNA bynding dyes.

(i) Amplifluor Universal Amplification and Detection System, IntergenCo., Purchase, N.Y.

In this system, PCR amplification and detection steps take place in thesame reaction vessel. Resultant PCR products fluoresce and can bemonitored with real-time or endpoint fluorescence detection instruments.The Amplifluor system is based on an innovative adaptation of themolecular beacon technology. Molecular beacons are hairpin-shapedoligonucleotides that contain fluorophore and quencher moieties.Molecular beacons act like switches that are normally closed to bringthe fluorophore/quencher pair together to turn fluorescence “off.” Whenprompted to undergo conformational changes that open the hairpinstructure, the fluorophore and quencher are separated, and fluorescenceis turned “on.” Similarly, the Amplifluor system uses a primer thatcontains a hairpin-shaped end in which fluorescein is paired up with thequencher 4-(dimethylamine)azo benzene sulfonic acid (DABSYL). However,Intergen points out that there is an important difference between theAmplifluor system and other currently available energy transfer-basedPCR methods (e.g., molecular beacons or Perkin-Elmer's Taqman™. InAmplifluor, the fluorescent oligonucleotides are actually incorporatedinto the reaction products. This enables the direct detection of PCRproducts, reducing the number of false positive reactions, which can becaused by even the most minimal carry-over contamination. Three primersare used to amplify products with Intergen's Amplifluor system. Forwardand reverse primers specific for the gene of interest are generated bythe user. Additionally, reactions contain the UniPrimer™Energy-Transfer-labeled Primer—the key component of the Amplifluorsystem. The 5′ end of UniPrimer consists of a hairpin structure labeledwith fluorescein and DABSYL. A tail sequence (Z) is at the primer's 3′end. The Z sequence acts as a universal PCR primer; it is specificallydesigned to reduce PCR background due to heterodimer formation. Any PCRreaction can be adapted to the Amplifluor system by synthesizing amodified version of one of the target-specific primers (the Z sequenceis simply added to the 5′ end of the modified primer). Conventionalpost-PCR detection methods such as gel electrophoresis or dot blottechniques are not required.

(ii) Molecular Beacon

The molecular beacon is a hairpin-shaped oligo with a loop sequencecomplementary to part of the target sequence and flanked by two armsthat anneal to form a short (5-7 base pair) stem. At the end of one armis a fluorophore and at the other a quencher that prevents fluorescencewhen the stem is intact. However, with careful consideration given tothe relative stability of the stem versus that of the beacon-targethybrid, the oligo is designed to remain folded in free solution but toreadily hybridize to any available target; once hybridized, the quencheris moved away from the fluorophore, which then fluoresces to signal thattarget is present. Molecular beacons thus can be used to monitorreal-time PCR by using a target sequence in the middle of the ampliconand measuring fluorescence during the annealing step of PCR.

In order to detect multiple targets in the same solution, molecularbeacons can be made in many different colors utilizing a broad range offluorophores. Dabcyl, a non-fluorescent chromophore, serves as theuniversal quencher for any fluorophore in molecular beacons. Owing totheir stem, the recognition of targets by molecular beacons is sospecific that single-nucleotide differences can be readily detected.Because of these properties, molecular beacons have been used for thedetection of RNAs within living cells, for monitoring the synthesis ofspecific nucleic acids in sealed reaction vessels, for homogenousone-tube assays for genotyping single-nucleotide variations in DNA andfor multiplex PCRs for the detection of four different pathogenicretroviruses (Vet et al., 1999).

When fully optimized, molecular beacons make for efficient detectionsystems, but occasionally some pitfalls are encountered. False positivesor low signal-to-background can result from impure preparations thatcontain free fluorophores or from oligos with a fluorophore but noquencher, or from design problems such as a stem that is too strong atlow temperatures. Care must be taken with design as well as with thenecessary control experiments to ensure that molecular beacons operateas intended.

(iii) TaqMan probe.

A cousin of the molecular beacon is the TaqMan probe from AppliedBiosystems of Foster City, Calif. This system exploits the 5′exonuclease activity of Taq DNA polymerase. During the PCR extension anannealed oligonucleotide that has a reporter fluorophore at the 5′exonuclease and a quencher at the 3′ exonuclease is chewed up by apolymerase 5′-3′ exonuclease activity, releasing the fluorophore fromits quencher (the presence of the TaqMan probe doesn't significantlyinhibit PCR product synthesis). The resulting fluorescence isproportional to the amount of PCR product.

(iv) The Dual Fluorophore

An alternative to the fluorophore-quencher system is a dual fluorophoreapproach that exploits FRET. This is the principle behind theLightCycler hybridization probes from Roche Molecular Biosystems ofIndianapolis. Two oligo probes, rather than TaqMan's one, anneal to theamplicon; one carries a fluorescein label (the FRET donor) at its 3′ endand the second is labeled with LC red 640 (the FRET acceptor) at its 5′end. The oligos are designed to hybridize in a head-to-tail orientationwith the fluorophores separated at a distance that is compatible withefficient energy transfer.

(v) Fluorescent Oligonucleotides for Homogeneous Detection, LifeTechnologies, Inc.

A novel fluorescent detection system that does not require a quenchingmoiety for homogeneous detection was developed. The technology is basedon oligonucleotides labeled with a single fluorophore with significantincrease in fluorescence intensity upon hybridization or incorporationinto double stranded DNA. This detection technology is a platform forfluorescent detection of nucleic acids in real time as well as in closedtube endpoint formats. This detection methodology has been used ashybridization probes and as amplification primers in homogenous PCRamplification assays.

(vi) SYBR Green I Dye

The fluorescent dye SYBR Green I binds to the minor groove of the DNAdouble helix. In solution, the unbound dye exhibits very littlefluorescence, however, fluorescence is greatly enhanced uponDNA-binding. SYBR Green I dye is very stable (only 6% of the activity islost during 30 amplification cycles).

At the beginning of amplification, the reaction mixture contains thedenatured DNA, the primers and the dye. The unbound dye molecules weaklyfluoresce, producing a minimal background fluorescence signal which issubtracted during computer analysis.

After annealing of the primers, a few dye molecules can bind to thedouble strand. DNA binding results in a dramatic increase of the SYBRGreen I molecules to emit light upon excitation.

During elongation, more and more dye molecules bind to the newlysynthesized DNA. If the reaction is monitored continuously, an increasein fluorescence is viewed in real-time. Upon denaturation of the DNA forthe next heating cycle, the dye molecules are released and thefluorescence signal falls.

Fluorescence measurement at the end of the elongation step of every PCRcycle is performed to monitor the increasing amount of amplified DNA. Toseparate specific from unspecific signals fluorescence can be measuredat high temperature. The unspecific products usually melt at a muchlower temperature than the specific product. Therefore, the specificityof the signal can be significantly enhanced if the temperature is raisednear to the melting point of the specific fragment

(vii) Other DNA Binding Dyes/Intercalators:

DNA binding dyes, some of which are intercalators, bind double-strandedDNA and to a lesser extent single-stranded DNA and RNA. With some ofthese dyes, binding to DNA substantially increases the intensity oftheir fluorescence. Dimeric dyes are noteworthy for their higheraffinity. RNA and single-stranded DNA stains can be used to detect RNAand single stranded DNA.

Other methods of detection are described in J. Ju et al., “Fluorescentenergy transfer dye-labeled primers for DNA sequence analysis,”Proceedings of the National Academy of Sciences, 92:4347-51, 1995; S.Tyagi, F. R. Kramer, “Molecular beacons: probes that fluoresce uponhybridization,” Nature Biotechnology, 14:303-8, 1996; A. J.-C. Eun,S.-M. Wong, “Molecular beacons: A new approach to plant virusdetection,” Phytopathology, 90:269-75, March 2000; L. G. Kostrikis etal., “Spectral genotyping of human alleles,” Science, 279:1228-19, 1998;G. Bonnet et al., “Thermodynamic basis of the enhanced specificity ofstructured DNA probes,” Proceedings of the National Academy of Sciences,96:6171-6, 1999; R. D. Oberst et al., “PCR-based DNA amplification andpresumptive detection of Escherichia coli O157:H7 with an internalfluorogenic probe and the 5′ nuclease (TaqMan) assay,” Applied andEnvironmental Microbiology, 64:3389-96, 1998; 1. Tapp et al.,“Homogenous scoring of single-nucleotide polymorphisms: comparison ofthe 5′-nuclease TaqMan assay and Molecular Beacon probes,”Biotechniques, 28:732-8, April 2000; I. A. Nazarenko et al., “A closedtube format for amplification and detection of DNA based on energytransfer,” Nucleic Acids Research, 25:2516-21, 1997; G. J. Nuovo et al.,“In situ amplification using universal energy transfer-labeled primers,”The Journal of Histochemistry and Cytochemistry, 47:273-9, 1999; D.Schuster, “Novel fluorescent oligonucleotides for homogenous detectionand quantitation of nucleic acids,” Abstracts from the CambridgeHealthcare Institute's fifth annual conference on Gene Quantification;Tyagi S and Kramer FR (1996) Molecular beacons: probes that fluoresceupon hybridization. Nat Biotechnol 14, 303-308; Tyagi S, Bratu D P, andKramer F R (1998) Multicolor molecular beacons for allelediscrimination. Nat Biotechnol 16, 49-53; Matsuo T (1998) In situvisualization of mRNA for basic fibroblast growth factor in livingcells. Biochimica Biophysica Acta 1379, 178-184; Sokol D L, Zhang X, LuP, and Gewirtz A M (1998) Real time detection of DNA-RNA hybridizationin living cells. Proc Natl Acad Sci USA 95, 11538-11543; Leone C, vanSchijndel H, van Gemen B, Kramer F R, and Schoen C D (1998) Molecularbeacon probes combined with amplification by NASBA enable homogeneous,real-time detection of RNA. Nucleic Acids Res 26, 2150-2155; Piatek A S,Tyagi S, Pol A C, Telenti A, Miller L P, Kramer F R, and Alland D (1998)Molecular beacon sequence analysis for detecting drug resistance inMycobacterium tuberculosis. Nat Biotechnol 16, 359-363; Kostrikis L G,Tyagi S, Mhlanga M M, Ho D D, and Kramer F R (1998) Spectral genotypingof human alleles. Science 279, 1228-1229; Giesendorf B A, Vet J A, TyagiS, Mensink E J, Trijbels F J, and Blom H J (1998) Molecular beacons: anew approach for semiautomated mutation analysis. Clin Chem 44, 482-486;Marras S A, Kramer F R, and Tyagi S (1999) Multiplex detection ofsingle-nucleotide variations using molecular beacons. Genet Anal 14,151-156; and Vet J A, Majithia A R, Marras S A, Tyagi S, Dube S, PoieszB J, and Kramer F R (1999) Multiplex detection of four pathogenicretroviruses using molecular beacons. Proc Natl Acad Sci USA 96,6394-6399.

VI. Quantitative Real-Time Polymerase Chain Reaction Using TaqMan

Quantitative real-time polymerase chain reaction using TaqMan and thePerkin-Elmer/Applied Biosystems division 7700 sequence detector (PE/ABD7700) provides an accurate method for determination of levels ofspecific DNA and RNA sequences in samples. It is based on detection of afluorescent signal produced proportionally during amplification of a PCRproduct.

Quantitative real-time PCR using the PE/ABD 7700 is based on detectionof a fluorescent signal produced proportionally during the amplificationof a PCR product. The chemistry is the key to the detection system. Aprobe is designed to anneal to the target sequence between thetraditional forward and reverse primers. The probe is labeled at the 5′end with a reporter fluorochrome (usually 6-carboxyfluorescein [6-FAM])and a quencher fluorochrome (6-carboxy-tetramethyl-rhodamine [TAMRA])added at any T position or at the 3′ end. The probe is designed to havea higher Tm than the primers, and during the extension phase, the probemust be 100% hybridized for success of the assay. As long as bothfluorochromes are on the probe, the quencher molecule stops allfluorescence by the reporter. However, as Taq polymerase extends theprimer, the intrinsic 5′ to 3′ nuclease activity of Taq degrades theprobe, releasing the reporter fluorochrome. The amount of fluorescencereleased during the amplification cycle is proportional to the amount ofproduct generated in each cycle.

The 7700 detection system consists of a 96-well thermal cycler connectedto a laser and charge-coupled device (CCD) optics system. An opticalfiber inserted through a lens is positioned over each well, and laserlight is directed through the fiber to excite the fluorochrome in thePCR solution. Emissions are sent through the fiber to the CCD camera,where they are analyzed by the software's algorithms. Collected data aresubsequently sent to the computer.

The sensitivity of detection allows acquisition of data when PCRamplification is still in the exponential phase. This is determined byidentifying the cycle number at which the reporter dye emissionintensities rises above background noise; this cycle number is calledthe threshold cycle (Ct). The Ct is determined at the most exponentialphase of the reaction and is more reliable than end-point measurementsof accumulated PCR products used by traditional PCR, methods. The Ct isinversely proportional to the copy number of the target template; thehigher the template concentration, the lower the threshold cyclemeasured.

There are many advantages to quantifying gene sequences using thistechnology, foremost being sensitivity and precision. This precisionexists because quantification of the gene sequence is determined by theCt, which is calculated during the exponential phase of the reaction.High specificity is conferred by the requirement of three oligos toanneal to the DNA before any data are collected.

Competitive PCR is another technique often used to quantify DNA or RNA.Optimization of competitive PCR is laborious and time consuming. Severaldilutions of target sequences must be tested to achieve a suitable ratioof target to competitor, and efficiencies of target and competitor mustbe similar. This assay is linear only over a very short range comparedwith quantification with the 7700. The number of samples that can beprocessed is also a limiting factor.

The applications for quantitative real-time PCR are innumerable.Detection of genomic or viral DNA in tissues can be a valuablediagnostic tool. Gene expression can be measured after extraction oftotal RNA and preparation of cDNA by a reverse transcription (RT) step.Setup and analysis are simple and can more easily be extended to theclinical environment than traditional PCR techniques.

Optimization of the PCR reaction is required for each primer and probeset. The optimal Mg2+ concentration is usually between 4 and 6 mM butsometimes can be as low as 2 mM. Optimal primer concentrations areusually between 100 and 800 nM. Optimization requires varying theconcentration of one primer relative to the other, because the optimalconcentration may not be the same for both. The optimal probeconcentration may be as low as 50 nM or as high as 200 nM. The optimalMg2+ concentration and reverse primer concentration must also bevalidated for the RT step.

The detection system is so sensitive that fewer than 10 copies of DNAcan be detected. Aerosol contamination of primers and probes is apotential problem if samples are prepared in the laboratory where DNA isbeing extracted.

For determination of pathogens, total nucleic acids are isolated. Aspecific cDNA can be produced by using the same reverse primer used inthe PCR reaction or by using random hexamer primers to produce a rangeof cDNA products. RNA can easily be prepared using kits such as RNAEasyfrom Qiagen (Valencia, Calif., USA) and Triazol from Life Technologies(Gaithersburg, Md., USA).

Multiplexing quantitative PCR reactions by using more than onefluorescent dye per tube became available for internal tube controls.Kits are available for 18S ribosomal RNA or forglyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a control. These twofluorochromes are preferred for use with FAM, the reporter used on theprobe.

If copy number is required, standard curves of plasmid DNA can beconstructed and assayed each time with samples containing the targetgene sequence. If the starting molecule is RNA, cRNA can be prepared andused as a standard. Kits are available to prepare RNA from plasmidscontaining the gene sequence. T7, T3, or SP6 primers typically are usedto prepare the cRNA. The cRNA produced must be validated in the RT andPCR reactions to determine if it is transcribed and amplified at thesame efficiency as the sample RNA present in a mixture of extractedRNAs.

Other important controls are no-amplification controls (NACs) andno-template controls (NTCs). NACs test for contamination of RNA bygenomic DNA. NTCs test for the contamination of assay reagents.

Several types of reaction mixes are available. The TaqMan Universal PCRMaster Mix, contains the core reagents in an easy to use 2× solution.The TaqMan Gold RT-PCR kit allows one-step or two-step RT-PCR. Theone-step option allows an investigator to set up the RT and PCR stepswithout opening the tube, whereas the two-step option separates the RTstep from the PCR. Master mixes can also be assembled by purchasing thevarious components, such as NTPs, buffer, Mg2+, and Taq polymerase, frommany other companies offering molecular biology reagents.

Primers and probes must be carefully designed. The Primer Expresssoftware, which is specifically designed to select the primers andprobes takes into account the required parameters for well-designedprimers and probe. These parameters include a Tm for the probe that is10° C. higher than the primers, primer Tms between 58° C. and 60° C.,amplicon size between 50 and 150 bases, absence of 5′ Gs, and primerlength.

The best design for primers and probes to use for the quantification ofRNA expression requires positioning of a primer or the probe in aconserved region of the virus, or in case of genetic testing, over anintron.

VII. Kits

In addition, the present invention also provides for a kit for use inconducting viral assays for efficacy that includes a mixture ofoligonucleotides, the mixture containing at least one the first primerand/or probe set that provides a detectable signal on the occurrence onthe transcription of viral nucleic acid; and at least one primer and/orprobe set provides a second detectable signal on the occurrence on thetranscription of host nucleic acid.

The present invention also provides for a kit for use in conductingtoxicity assays for efficacy that includes a mixture ofoligonucleotides, the mixture containing at least one the first primerand/or probe that provides a detectable signal on the occurrence on thetranscription of host mitochondrial nucleic acid; and at least oneprimer and/or probe provides a second detectable signal on theoccurrence on the transcription of host nuclear nucleic acid.

In particular, the kit comprises a primer/probe set for host nucleicacid wherein the primers are given by Sequence ID No. 1 and 2, and theprobe is a sequence given by Sequence ID No. 3 along with a fluorescentdye and quenching dye.

In particular, the kit comprises a primer/probe set for viral nucleicacid for HIV-1 wherein the primers are given by Sequence ID No. 4 and 5,and the probe is a sequence given by Sequence ID No. 6 along with afluorescent dye and quenching dye.

In particular, the kit comprises a primer/probe set for viral nucleicacid for HCV wherein the primers are given by Sequence ID No. 7 and 8,and the probe is a sequence given by Sequence JD No. 9 along with afluorescent dye and quenching dye.

In particular, the kit comprises a primer/probe set for viral nucleicacid for BVDV wherein the primers are given by Sequence ID No. 10 and11, and the probe is a sequence given by Sequence ID No. 12 along with afluorescent dye and quenching dye.

In particular, the kit comprises a primer/probe set for viral nucleicacid for HBV wherein the primers are given by Sequence ID No. 13 and 14,and the probe is a sequence given by Sequence ID No. 5 along with afluorescent dye and quenching dye.

In particular, the kit comprises a primer/probe set for viral nucleicacid for RSV wherein the primers are given by Sequence ID No. 16 and 17,and the probe is a sequence given by Sequence ID No. 18 along with afluorescent dye and quenching dye.

In particular, the kit comprises a primer/probe set for hostmitochondiral nucleic acid wherein the primers are given by Sequence IDNo. 19 and 20, and the probe is a sequence given by Sequence ID No. 21along with a fluorescent dye and quenching dye.

This invention is further illustrated in the following sections. Theexamples contained therein are set forth to aid in an understanding ofthe invention. The following examples are illustrative of the processesand products of the present invention; but this section is not intendedto, and should not be interpreted to, limit in any way the invention setforth in the claims that follow thereafter. Equivalent, similar, orsuitable solvents, reagents or reaction conditions may be substitutedfor those particular solvents, reagents or reaction conditions describedherein without departing from the general scope of the method.

EXAMPLES Example 1

HIV-1 Cell culture

Human PBMC (1×10⁶ cells/T25 flask) were PHA stimulated for 2 days, andinfected with either a sensitive (xxBRU) or a 3TC-resistant (184V) HIV-1strain at 100 TCID₅₀. The culture was kept for 5 days in presence oftest antiviral compounds at serial 1-log dilutions. Subsequently, humanPBMC were removed from the culture supernatant by centrifugation (10min, 400×g, 4° C.). This clarified supernatant was tested either in theRT-assay, or in the real-time RT-PCR assay.

Example 2

Reverse Transcriptase (RT) Assay

Virus particles present in a 1 mL aliquot of culture supernatant wereconcentrated by centrifugation (2 hr, 20,000×g, 4° C.). After the 2 hourspin, supernatant fluid was removed completely and the virus pellet wasdispensed into a 100 μL Virus Solubilization Buffer (VSB: 0.5% TritonX-100; 0.8 M NaCl, 0.5 mM phenylmethylsulfonyl, 20% glycerol, 50 mMTris.HCl pH 7.8). A 10 μL aliquot of RT-VSB was mixed with 75 μL RTcocktail (60 mM Tris.HCl pH 7.8, 12 mM MgCl₂, 6 mM DTT, 6 μg/mL Poly(rA)-Poly (dT), 1.2 mM dATP, and 80 μCi/mL H³-TTP) and incubated for 2hr at 37° C. Subsequently 100 μL of 10% TCA was added, and the totalamount of incorporated H³-TTP was counted.

Example 3

RT-PCR Primer and Probe Assessment

The TaqMan probe and primers were designed by using the Primer Expresssoftware (Applied Biosystems, CA) and are covering highly conservedsequences complementary to the DNA sequences present in HIV-1 RNA. Byscanning the different genotypes of group M for regions containing onlyminor variability, the conserved domain was discovered. As a result, theregion in the HIV-1 RT domain between codon 200 and 280 fulfilled therequired criteria; thus this region was used to design an appropriateset of primers and probe that could work in real time PCR (“RT-PCR”).Primer sequences are as follows: sense 5′-TGGGTTATGAACTCCATCCTGAT-3′(Sequence ID No.) and 5,-TGTCATTGACAGTCCAGCTGTCT-3′ (Sequence ID No.);the probe sequence is 5′-fluoresentdye-TTTCTGGCAGCACTATAGGCTGTACTGTCCATT-quenching dye-3′ (Sequence IDNo.). In this particular case, the probe was labeled with FAM at the 5′end, and the quencher molecule is TAMARA, provided at the 3′ end. Anyother combination of reporter and quencher dyes can be used as well.

The primer and probe set gave a linear range over 6 logs when tested onserial 1-log dilutions of cultured virus. In order to evaluate thisprimer/probe set with an FDA approved methodology for viral loadmeasurement, a 1-log dilution series of a clinical HIV-1 genotype βisolate (attenuated in vitro to obtain a high viral load) was tested byreal time RT-PCR and by Roche Amplicor HIV-1 Monitor (FIG. 1). In thisexperiment, the 10⁻⁶ diluted sample became positive at threshold cycle(Ct=35.52), which corresponded with a 1410 copies/mL in the Rochemonitor HIV-1 version II assay. When validated over a dynamic range of 3logs of virus, there was perfect correlation between the twomethodologies (FIG. 1) with a lower limit of detection for the real-timeRT-PCR assay of 141 copies/mL (Ct=38.85).

Example 4

Real-time RT-PCR Assay

The real-time RT-PCR technology was evaluated against the NASBA HIV-1viral load assay. HIV-1 nucleic acid sequences was amplified using thedesigned probes and primers as described above. Viral RNA present in theculture supernatant was prepared using commercially available columns(QIAamp Viral RNA mini Kit, Qiagen, CA). The amplification reactionmixture was incubated for two minutes at 50° C., then ten minutes at 95°C. Then, the mixture was amplified using forty cycles of a two-stepamplification reaction at 95° C. for fifteen seconds then sixty secondsat 60° C. Real-time RT-PCR-amplified RNA was detected in real-time bymonitoring increases in fluorescence signal that resulted fromdegradation of a quenched fluorescent probe molecule following to thehybridization of the probe to the amplified viral DNA (TaqMan 7700chemistry, Applied Biosystems, CA).

A total of 5 μL RNA was RT-amplified using reagents and conditions asdescribed by the manufacturer (Applied Biosystems, CA). The standardcurve ranged from 1.41×10² copies/mL to over 1.41×10⁸ copies/mL. Copynumbers were calibrated using the Roche Amplicor HIV-1 Monitor test™(Roche Diagnostics, Branchburg, N.J.), or the NASBA HIV-1 viral loadassay (Organon Technika).

Samples containing HIV-1 (genotype B) over a range of 3 logs (5×10³ to5×10⁶ copies/mL) were tested in both methodologies. The correlationbetween the two methodologies is shown in FIG. 2. All samples testedfelt within the 95% confidence interval, and only 2 samples were outsidethe 99% confidence interval. It can be concluded that the currentlydesigned primer and probe set allowed reliable quantification of theboth clinical samples and HIV-1 in vitro virus preparations. Thereal-time-RT-PCR has a lower limit of detection of 141 copies/mL andshowed linearity over 6-logs of virus dilution.

Example 5

Optimization

Optimization of the PCR reaction is required for each primer and probeset. The optimal Mg2+ concentration is usually between 4 and 6 mM butsometimes can be as low as 2 mM. Optimal primer concentrations areusually between 100 and 800 nM. Optimization requires varying theconcentration of one primer relative to the other, because the optimalconcentration may not be the same for both. The optimal probeconcentration may be as low as 50 nM or as high as 200 nM. The optimalMg2+ concentration and reverse primer concentration must also bevalidated for the RT step.

Potential Contamination

The detection system is so sensitive that fewer than 10 copies of DNAcan be detected. Aerosol contamination of primers and probes is apotential problem if samples are prepared in the laboratory where DNA isbeing extracted.

Sample Preparation

For determination of pathogens, total nucleic acids are isolated. Aspecific cDNA can be produced by using the same reverse primer used inthe PCR reaction or by using random hexamer primers to produce a rangeof cDNA products. RNA can easily be prepared using kits such as RNAEasyfrom Qiagen (Valencia, Calif., USA) and Triazol from Life Technologies(Gaithersburg, Md., USA).

Controls

Multiplexing quantitative PCR reactions by using more than onefluorescent dye per tube became available for internal tube controls.Kits are available for 18S ribosomal RNA or forglyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a control. These twofluorochromes are preferred for use with FAM, the reporter used on theprobe.

If copy number is required, standard curves of plasmid DNA can beconstructed and assayed each time with samples containing the targetgene sequence. If the starting molecule is RNA, CRNA can be prepared andused as a standard. Kits are available to prepare RNA from plasmidscontaining the gene sequence. T7, T3, or SP6 primers typically are usedto prepare the cRNA. The cRNA produced must be validated in the RT andPCR reactions to determine if it is transcribed and amplified at thesame efficiency as the sample RNA present in a mixture of extractedRNAs.

Other important controls are no-amplification controls (NACs) andno-template controls (NTCs). NACs test for contamination of RNA bygenomic DNA. NTCs test for the contamination of assay reagents.

Reaction Mix

Several types of reaction mixes are available. The TaqMan Universal PCRMaster Mix, contains the core reagents in an easy to use 2× solution.The TaqMan Gold RT-PCR kit allows one-step or two-step RT-PCR. Theone-step option allows an investigator to set up the RT and PCR stepswithout opening the tube, whereas the two-step option separates the RTstep from the PCR. Master mixes can also be assembled by purchasing thevarious components, such as NTPs, buffer, Mg2+, and Taq polymerase, frommany other companies offering molecular biology reagents.

Primer and Probe Design

Primers and probes must be carefully designed. The Primer Expresssoftware, which is specifically designed to select the primers andprobes takes into account the required parameters for well-designedprimers and probe. These parameters include a Tm for the probe that is10° C. higher than the primers, primer Tms between 58° C. and 60° C.,amplicon size between 50 and 150 bases, absence of 5′ Gs, and primerlength.

The best design for primers and probes to use for the quantification ofRNA expression requires positioning of a primer or the probe in aconserved region of the virus, or in case of genetic testing, over anintron.

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Markowitz 1999. Use ofreal-time PCR and molecular beacons to detect virus replication in humanimmunodeficiency virus type 1-infected individuals on prolongedeffective antiretroviral therapy. J Virol. 73:6099-6103; Locatelli, G.,F. Santoro, F. Veglia, A. Gobbi, P. Lusso, and M. S. Malnati 2000.Real-time quantitative PCR for human herpesvirus 6 DNA. J ClinMicrobiol. 37:4042-4048; Machida, U., M. Kami, T. Fukui, Y. Kazuyama, M.Kinoshita, Y. Tanaka, Y. Kanda, S. Ogawa, H. Honda, S. Chiba, K. Mitani,Y. Muto, K. Osumi, S. Kimura, and H. Hirai 2000. Real-time automated PCRfor early diagnosis and monitoring of cytomegalovirus infection afterbone marrow transplantation. J Clin Microbiol. 38:2536-2542; Martell,M., J. Gomez, J. I. Esteban, S. Sauleda, J. Quer, B. Cabot, R. Esteban,and J. Guardia 1999. High-throughput real-time reverse transcription-PCRquantitation of hepatitis C virus RNA. J Clin Microbiol. 37:327-332;Najioullah, F., D. Thouvenot, and B. Lina 2001. Development of areal-time PCR procedure including an internal control for themeasurement of HCMV viral load. J Virol Methods. 92:55-64.; Nicoll, S.,A. Brass, and H. A. Cubie 2001. Detection of herpes viruses in clinicalsamples using real-time PCR. J Virol Methods. 96:25-31; Niesters, H. G.,J. van Esser, E. Fries, K. C. Wolthers, J. Comelissen, and A. D.Osterhaus 2000. Development of a real-time quantitative assay fordetection of epstein-barr virus. J Clin Microbiol. 38:712-715; Nitsche,A., N. Steuer, C. A. Schmidt, O. Landt, H. Ellerbrok, G. Pauli, and W.Siegert 2000. Detection of human cytomegalovirus DNA by real-timequantitative PCR. J Clin Microbiol. 38:2734-2737; Ohyashiki, J. H., A.Suzuki, K. Aritaki, A. Nagate, N. Shoji, K. Ohyashiki, T. Ojima, K. Abe,and K. Yamamoto 2000. Use of real-time PCR to monitor human herpesvirus6 reactivation after allogeneic bone marrow transplantation. Int J MolMed. 6:427-432.; Pevenstein, S. R., R. K. Williams, D. McChesney, E. K.Mont, J. E. Smialek, and S. E. Straus 1999. Quantitation of latentvaricella-zoster virus and herpes simplex virus genomes in humantrigeminal ganglia. J Virol. 73:10514-10548; Ratge, D., B. Scheiblhuber,M. Nitsche, and C. Knabbe 2000. High-speed detection of blood-bornehepatitis C virus RNA by single-tube real-time fluorescence reversetranscription-PCR with the LightCycler. Clin Chem. 46:1987-1989; Saha,B. K., B. Tian, and R. P. Bucy 2001. Quantitation of HIV-1 by real-timePCR with a unique fluorogenic probe J Virol Methods. 93:33-42; Sauleda,S., H. J. Reesink, J. I. Esteban, G. Hess, R. Esteban, and J. Guardia1999. Profiles of GBV-C/hepatitis G virus markers in patients coinfectedwith hepatitis C virus. J Med Virol. 59:45-51; Schutten, M., B. van denHoogen, M. E. van der Ende, R. A. Gruters, A. D. Osterhaus, and H. G.Niesters 2000. Development of a real-time quantitative RT-PCR for thedetection of HIV-2 RNA in plasma. J Virol Methods. 88:81-87; Takeuchi,T., A. Katsume, T. Tanaka, A. Abe, K. Inoue, K. Tsukiyama-Kohara, R.Kawaguchi, S. Tanaka, and M. Kohara 1999. Real-time detection system forquantification of hepatitis C virus genome. Gastroenterology.116:636-642; Tanaka, N., H. Kimura, K. Iida, Y. Saito, I. Tsuge, A.Yoshimi, T. Matsuyama, and T. Morishima 2000. Quantitative analysis ofcytomegalovirus load using a real-time PCR assay. J Med Virol.60:455-462; Tucker, R. A., E. R. Unger, B. P. Holloway, and D. C. Swan2001. Real-time PCR-based fluorescent assay for quantitation of humanpapillomavirus types 6, 11, 16, and 18. Mol Diagn. 6:39-47; Tyagi, S.,and F. R. Kramer 1996. Molecular beacons: probes that fluoresce uponhybridization. Nat Biotechnol. 14:303-308; van Elden, L. J., M. Nijhuis,P. Schipper, R. Schuurman, and A. M. van Loon 2001. Simultaneousdetection of influenza viruses A and B using real-time quantitative PCR.J Clin Microbiol. 39:196-200; Vet, J. A., A. R. Majithia, S. A.Majithia, S. Tyagi, S. Dube, B. J. Poiesz, and F. R. Kramer 1999.Multiplex detection of four pathogenic retroviruses using molecularbeacons. Proc Natl Acad Sci USA. 96:6394-6399.; Wagner, H. J., W. Jabs,F. Smets, M. Wessel, L. Fischer, G. Offner, H. Kirchner, and P. Bucsky2000. Real-time polymerase chain reaction (RQ-PCR) for the monitoring ofEpstein-Barr virus (EBV) load in peripheral blood mononuclear cells.Klin Padiatr. 212:206-210; Walker, N. J. 2001. Real-time andquantitative PCR: applications to mechanism-based toxicology. J BiochemMol Toxicol. 15:121-127; White, I. E., and T. B. Campbell 2000.Quantitation of cell-free and cell-associated Kaposi'ssarcoma-associated herpesvirus DNA by real-time PCR. J Clin Microbiol.38:1992-1995;

Example 6

Real-Time RT-PCR Assay for HIV-1.

HIV-1 particles were brought into culture using human PBM cells. ViralRNA present in the culture supernatant was prepared using commerciallyavailable columns (QIAamp Viral RNA mini Kit, Qiagen, CA).RT-PCR-amplified RNA was detected in real-time by monitoring increasesin fluorescence signal. A total of 5 L RNA was RT-amplified usingreagents and conditions as described by the manufacturer (AppliedBiosystems, CA). The standard curve ranged from 1.41×10² copies/mL toover 1.41×10⁸ copies/mL. Copy numbers were calibrated using the RocheAmplicor HIV-1 Monitor test™ (Roche Diagnostics, Branchburg, N.J.), orthe NASBA HIV-1 viral load assay (Organon Technika). Correlationcoefficient is in all experiments greater than 0.99. (FIG. 1).

Example 7

Real-Time RT-PCR assay for HCV.

As of today, the only reliable and available system for HCV RNAreplication is the replicon system in Huh7 cells. The cells were broughtinto culture for several days and total RNA present in the culture wasprepared using commercially available columns (QIAamp Viral RNA miniKit, Qiagen, CA). RT-PCR-amplified RNA was detected in real-time bymonitoring increases in fluorescence signal. A total of 5 L RNA wasRT-amplified using reagents and conditions as described by themanufacturer (Applied Biosystems, CA). The standard curve ranged from 45IU/mL to over 4.7×10⁷ IU/mL. Copy numbers were calibrated using theRoche Amplicor HCV Monitor test™ (Roche Diagnostics, Branchburg, N.J.).Correlation coefficient is in all experiments greater than 0.99. (FIG.2).

Example 8

Real-Time RT-PCR Assay for HBV.

HBV viral particles are released from at leasts three different celllines: HepG2.2.1.5, HEPAD38 and HepAD79 cell lines. The cells werebrought into culture for several days and total nucleic acids present inthe culture supernatant, or in the cells, was prepared usingcommercially available columns (QIAamp Viral RNA mini Kit, Qiagen, CA).PCR-amplified DNA was detected in real-time by monitoring increases influorescence signal. A total of 5 L DNA was RT-amplified using reagentsand conditions as described by the manufacturer (Applied Biosystems,CA). The standard curve ranged from 2 copies to over 2×10⁷ copies perreaction mix. Copy numbers were calculated form OD260 values obtainedfrom an HBV standard. Correlation coefficient is in all experimentsgreater than 0.99.

Example 9

Real-Time RT-PCR Assay for BVDV

BVDV viral particles are released from infection experiments using thestrain NADL on MDBK cells (both available form ATTC). After infection,the cell were brought into culture for several days and total nucleicacids present in the culture supernatant, or in the cells, was preparedusing commercially available columns (QIAamp Viral RNA mini Kit, Qiagen,CA). RT-PCR-amplified RNA was detected in real-time by monitoringincreases in fluorescence signal. A total of 5 L DNA was RT-amplifiedusing reagents and conditions as described by the manufacturer (AppliedBiosystems, CA). The standard curve ranged from 0.6 plaque forming unitsto over 6×10³ plaque forming units per reaction mix. Plaque formingunits were calculated form traditional plaque assays. Correlationcoefficient is in all experiments greater than 0.99.

Example 10

Real-Time RT-PCR Assay for RSV

RSV viral particles are released from infection experiments using theavailable virus strain derived from a clinical sample on A549 or Hep2cells. After infection, the cell were brought into culture for severaldays and total nucleic acids present in the culture supernatant, or inthe cells, was prepared using commercially available columns (QIAampViral RNA mini Kit, Qiagen, CA). RT-PCR-amplified RNA was detected inreal-time by monitoring increases in fluorescence signal. A total of 5 LDNA was RT-amplified using reagents and conditions as described by themanufacturer (Applied Biosystems, CA). The standard curve ranged from 70plaque forming units to over 7×110 plaque forming units/mL. Plaqueforming units were calculated form traditional plaque assays.Correlation coefficient is in all experiments greater than 0.99. Hep2cells gave the highest virus titer after 72 hours of incubation, theamount of cells used varied between 10,000 and 50,000 cells per well,but there were no differences observed in total amount of virusproduction at 72 hours.

Example 11

Real-Time PCR Assay for Human Mitochondrial DNA and β-actin DNA

The current inventions involve the amplification of these two genetictargets for mitochondrial toxicity testing. Therefore, a set of primersand fluorescent probes for both mitochondrial and nuclear DNA or RNA wasdesigned.

As one illustration of this method, in a first step, HepG2 cells arekept in culture in presence of 10 microMolar of a set of candidateantiviral agents. Subsequently, total DNA is isolated from culturedHepG2 cells by means of a commercially available columns (QIAamp DNABlood Mini Kit, Qiagen, CA). Total DNA was eluted from columns in 200 Lwater. The mitochondrial gene and nuclear gene are then amplified with aquantitative real-time PCR protocol using the suitable primers andprobes. Reagents and conditions used in quantitative PCR were purchasedfrom PE-Applied Biosystems.

In a separate experiment, the amplification efficiencies of both targetswere evaluated. The standard curve that was created using the dilutedtotal cell DNA showed linearity over 4 logs [FIG. 3]. Furthermore, FIG.3 demonstrates that efficiencies of target and reference amplificationare approximately equal, because the value of the slope of input amountversus DeltaCt (Ct β-actin-Ct mitochondrial; Ct=PCR cycle thresholdwhere a sample becomes detectable) is less than 0.1.

There are at least two methods to obtain accurate quantity measurements,one method is using standard curves, the other method is known as thecomparative cycle threshold method. The basics of the two methods areexplained in the User Bulletin # 2 of PE Applied Biosystems. Since bothtarget mitochondrial and the nuclear endogenous control gene areamplified with almost identical efficiencies using the describedprimer-probe sets, either method can be used to measure themitochondrial toxicities induced by antiviral agents. Preferably, thecomparative Ct method is used. This method uses arithmetic formulas toachieve the same result for relative quantification as obtained bystandard curve methods (see

User Bulletin #2; PE Applied Biosystems). In this arithmetic formula,the amount of target (mitochondrial DNA) is normalized to a calibrator(nuclear gene) and is relative to an endogenous reference (no drugcontrol at day 7 or 14, depending on the setup of the experiment). Thisarithmetic formula is given by 2^(−Ct).

In order to find out whether antiviral compounds should have anyinhibitory effect on the mitochondrial DNA polymerase γ themitochondrial COXII gene and the nuclear -actin gene were amplifiedsimultaneously. The relative mitochondrial DNA polymerase γ toxicity oftwo antiviral compounds (−)-FTC and D-DDC were compared with somecandidate new antiviral compounds. FIG. 4 demonstrates the resultsobtained for these antiviral agents. It is clear from this figure that(−)-FTC does not induce any significant mitochondrial DNA reduction ascompared to the no-drug control. Instead, important differences wereobserved for D-DDC at 1 and 10 microM concentration. The observedreduction in mitochondrial DNA in the DDC settings illustrates theusefulness of the simultaneous amplification of two or more differenttargets in molecular toxicology.

Similar results were also obtained if this technology was carried out ina quantitative reverse-transcriptase-PCR protocol. This approachmeasures the potential inhibition of antiviral compounds for themitochondrial RNA polymerase, in comparison with the nuclear RNApolymerases I (generating mainly rRNA transcripts), RNA polymerase II(generating mainly mRNA transcripts), or of lesser importance, RNApolymerase III (generating mainly tRNA transcripts). To obtain suchresults, amplification of either rRNA, or -actin mRNA as calibrator isrequired. In these experiments and after calibration against therelevant nuclear RNA polymerase transcripts and normalization for notreatment, DDC also showed a significant reduction in mitochondrial RNAlevels, while (−) FTC did not affect the COXII RNA levels.

This approach can be used to evaluate the molecular toxicity levels ofany candidate antiviral compounds tested in any cell type.

Total DNA is isolated from cultured HepG2 cells by commerciallyavailable columns (QIAamp DNA Blood Mini Kit, Qiagen, CA). Total DNA waseluted from columns in 200 μL of water. The mitochondrial gene andnuclear gene are then amplified with a quantitative real-time PCRprotocol using suitable primers and probes. A set of primers andfluorescent probes for both nuclear and mitochondrial DNA or RNA wasdesigned; the endogenous control DNA primer set is given by 5′-GCG CGGCTA CAG CTT CA-3′ (Sequence ID No.) and 5′-TCT CCT TAA TGT CAC GCA CGAT-3′ (Sequence ID No.); the mitochondrial DNA primer set is given by5′-TGC CCG CCA TCA TCC TA-3′ (Sequence ID No.) and 5′-TCG TCT GTT ATGTAA AGG ATG CGT-3′ (Sequence ID No.). The probe for nuclear gene isgiven by 5′-fluorescent Dye-CAC CAC GGC CGA GCG GGA-fluorescentquencher-3′ (Sequence ID No.); fluorescent labeled probes formitochondrial genome is given by 5′-fluorescent Dye-TCC TCA TCG CCC TCCCAT CCC-fluorescent quencher-3′ (Sequence ID No.). Reagents andconditions used in quantitative PCR were purchased from PE-AppliedBiosystems.

The standard curve created using the diluted total cell DNA showedlinearity over 4 logs [FIG. 4]. Furthermore, FIG. 4 demonstrates thatefficiencies of target and reference amplification are approximatelyequal, because the value of the slope of input amount versus ΔCt (Ctβ-actin-Ct mitochondrial; Ct=PCR cycle threshold where a sample becomesdetectable) is less than 0.1.

There are at least two methods to obtain accurate quantity measurements,one method is using standard curves, and the other method is known asthe comparative cycle threshold method. The basics of the two methodsare explained in the User Bulletin # 2 of PE Applied Biosystems. Sinceboth target mitochondrial and the nuclear endogenous control gene areamplified with almost identical efficiencies using the describedprimer-probe sets, either method can be used to measure themitochondrial toxicities induced by antiviral agents. Preferably, thecomparative Ct method is used. This method uses arithmetic formulas toachieve the same result for relative quantification as obtained bystandard curve methods (see User Bulletin #2; PE Applied Biosystems). Inthis arithmetic formula, the amount of target (mitochondrial DNA) isnormalized to an endogenous reference (nuclear gene) and is relative toa calibrator (no drug control at day 7 or 14, depending on the setup ofthe experiment). This arithmetic formula is given by 2^(−ΔΔCt).

Example 12

Simultaneous Amplification of HCV RNA and Cellular Targets.

Huh7 cells harboring the HCV replicon can be cultivated in DMEM media(high glucose, no pyruvate) containing 10% fetal bovine serum, 1×non-essential Amino Acids, Pen-Strep-Glu (100 units/liter, 100microgram/liter, and 2.92 mg/liter, respectively) and 500 to 1000microgram/milliliter G418. Antiviral screening assays can be done in thesame media without G418 as follows: in order to keep cells inlogarithmic growth phase, seed cells in a 96-well plate at low density,for example 1000 cells per well. Add the test compound immediate afterseeding the cells and incubate for a period of 3 to 7 days at 37° C. inan incubator. Media is then removed, and the cells are prepared fortotal nucleic acid extraction (including replicon RNA and host RNA).Replicon RNA can then be amplified in a Q-RT-PCR protocol, andquantified accordingly. The observed differences in quantification ofreplicon RNA is one way to express the antiviral potency of the testcompound. A typical experiment demonstrates that in the negative controland in the non-active compounds-settings a comparable amount of repliconis produced. This can be concluded because the measured threshold-cyclefor HCV RT-PCR in both setting is close to each other. In suchexperiments, one way to express the antiviral effectiveness of acompound is to subtract the threshold RT-PCR cycle of the test compoundwith the average threshold RT-PCR cycle of the negative control. Thisvalue is called DeltaCt (Ct). A Ct of 3.3 equals a 1-log reduction(equals ECO) in replicon production. Compounds that result in areduction of HCV replicon RNA levels of greater than 2 Ct values (75%reduction of replicon RNA) are candidate compounds for antiviraltherapy. However, this HCV Ct value does not include any specificityparameter for the replicon encoded viral RNA-dependent RNA polymerase.In a typical setting, a compound might reduce both the host RNApolymerase activity and the replicon-encoded polymerase activity.Therefore, quantification of rRNA (or any other host RNA polymerase Iproduct) or beta-actin mRNA (or any other host RNA polymerase II) andcomparison with RNA levels of the no-drug control is a relativemeasurement of the effect of the test compound on host RNA polymerases.

With the availability of both the HCV ΔCt data and the rRNA ΔCt, aspecificity parameter can be introduced. This parameter is obtained bysubtracting both ΔCt values from each other. This results in ΔΔCtvalues; a value above 0 means that there is more inhibitory effect onthe replicon encoded polymerase, a ΔΔCt value below 0 means that thehost rRNA levels are more affected than the replicon levels. As anillustration of this technology, the antiviral activity of testedcompounds, expressed as ΔΔCt values, is given in FIG. 5. As a generalrule, ΔΔCt values above 2 are considered as significantly different fromthe no-drug treatment control, and hence, is an interested compound forfurther evaluation. However, compounds with a ΔΔCt value of less than 2,but showing limited molecular cytotoxicty data (rRNA ΔCT between 0 and2) are also possible active candidate compounds for further evaluation

In another typical setting, a compound might reduce the host RNApolymerase activity, but not the host DNA polymerase activity.Therefore, quantification of rDNA or beta-actin DNA (or any other hostDNA fragment) and comparison with DNA levels of the no-drug control is arelative measurement of the inhibitory effect of the test compound oncellular DNA polymerases. With the availability of both the HCV ΔCt dataand the rDNA ΔCt, a specificity parameter can be introduced. Thisparameter is obtained by subtracting both ΔCt values from each other.This results in ΔΔCt values; a value above 0 means that there is moreinhibitory effect on the replicon encoded polymerase, a ΔΔCt value below0 means that the host rDNA levels are more affected than the repliconlevels. As a general rule, ΔΔCt values above 2 are considered assignificantly different from the no-drug treatment control, and hence,is an interested compound for further evaluation. However, compoundswith a ΔΔCt value of less than 2, but with limited molecular cytotoxicty(rDNA ΔCT between 0 and 2) are also possible active candidate compoundsfor further evaluation

Quantitative real-time PCR antiviral screening can be combined withcalibration for a nuclear RNA targets (in RT-PCR) in the followingsettings: anti-HCV compound screening can be combined with rRNAcalibration, or -actin mRNA calibration, or any other nuclear ormitochondrial gene calibration. Anti-HIV compound screening can becombined with rRNA calibration, -actin mRNA calibration or any othernuclear or mitochondrial gene calibration. Anti-HBV compound screeningcan be combined with rRNA calibration, -actin mRNA calibration, or anyother nuclear or mitochondrial gene calibration. Anti-RSV compoundscreening can be combined with rRNA calibration, -actin mRNAcalibration, or any other nuclear or mitochondrial gene calibration.Anti-BVDV compound screening can be combined with rRNA calibration,-actin mRNA calibration or any other nuclear or mitochondrial genecalibration. Anti-lentivirus compound screening can be combined withrRNA calibration, -actin mRNA calibration or any other nuclear ormitochondrial gene calibration. Anti-flaviviridae (Flavivirus,Hepacivirus, Pestivirus) compound screening can be combined with rRNAcalibration, -actin mRNA calibration or any other nuclear ormitochondrial gene calibration. Anti-hepadnavirus compound screening canbe combined with rRNA calibration, -actin mRNA calibration or any othernuclear or mitochondrial gene calibration. Anti-Picornavirus compoundscreening can be combined with rRNA calibration, -actin mRNA calibrationor any other nuclear or mitochondrial gene calibration.Anti-Herpetoviridae (HSV, HCMV, EBV) compound screening can be combinedwith rRNA calibration, -actin mRNA calibration or any other nuclear ormitochondrial gene calibration.

Quantitative real-time PCR antiviral screening can be combined withcalibration for a nuclear DNA target (in PCR) in the followingconditions. anti-HCV compound screening can be combined with rDNAcalibration, or -actin DNA calibration, or any other nuclear ormitochondrial gene calibration. Anti-HIV compound screening can becombined with rDNA calibration, -actin DNA calibration or any othernuclear or mitochondrial gene calibration. Anti-HBV compound screeningcan be combined with rDNA calibration, -actin DNA calibration or anyother nuclear or mitochondrial gene calibration. Anti-RSV compoundscreening can be combined with rDNA calibration, -actin DNA calibrationor any other nuclear or mitochondrial gene calibration. Anti-BVDVcompound screening can be combined with rDNA calibration, -actin DNAcalibration, or any other nuclear or mitochondrial gene calibration.Anti-lentivirus compound screening can be combined with rDNAcalibration, -actin DNA calibration or any other nuclear ormitochondrial gene calibration. Anti-flaviviridae (Flavivirus,Hepacivirus, Pestivirus) compound screening can be combined with rDNAcalibration, -actin DNA calibration or any other nuclear ormitochondrial gene calibration. Anti-hepadnavirus compound screening canbe combined with rDNA calibration, -actin DNA calibration or any othernuclear or mitochondrial gene calibration. Anti-Picornavirus compoundscreening can be combined with rDNA calibration, -actin DNA calibrationor any other nuclear or mitochondrial gene calibration.

Anti-Herpetoviridae (HSV, HCMV, EBV) compound screening can be combinedwith rDNA calibration, -actin DNA calibration or any other nuclear ormitochondrial gene calibration.

Example 13

Toxicity Assays

HepG2, VERO (5×10³ cells per well), CEM (2.5×10³ per well), and PBMC(5×10⁴ per well) were seeded in 96-well plates at in the presence ofincreasing concentrations of the test compound and incubated in a 37°C., 5% CO₂ incubator. After a three day-incubation, or 4 for CEM, or 5days for PBMC, cell viability and mitochondrial activity were measuredin a colorimetric assay using the MTS- or MTT dye (Promega, Wis.).

Example 14

Antiviral RT-PCR Versus RT Assay

β-L and β-D analogues of2′,3′-didehydro-2′,3′-dideoxy-2′-fluoro-4′-thio-cytidine[“d4-2′-F-(4S-pentenyl)-C”] were compared with a selection of antiviralsthat are currently FDA-approved, or in clinical trial such as AZT, 3TC,d4T, and (−)-FTC against a two HIV-1 viral strains a sensitive strain,xxBRU, and a 3TC-resistant viral strain with the 184V mutation. HumanPBMC were PHA stimulated for 2 days, HIV-1 infected, and kept in culturefor 5 days in presence of test compounds at different concentrations.Subsequently, culture supernatant was clarified, and tested for reversetranscription activity by two separate methods. The first method is thestandard endogenous viral RT assay with read-out in log counts perminute/mL (CPM/mL) by incorporating tritium-labeled TTP; the second isthe RT-PCR method disclosed herein, a quantification method of HIV-1viral load using real-time PCR quantification assay with read-out in logcopies/mL. FIG. 3 shows the result for some of the tested compound onboth viral strains. Although the two methodologies are measuring fordifferent items (viral RNA versus active RT enzyme) results werenot-significantly different from each other (FIG. 3, Table 1). Themedian 50% (EC₅₀) and 90% (EC₉₀) effective antiviral concentrations werein concordance for the two methodologies used.

Wild type xxBRU virus production in this system was very high, with atotal of up to 3×10⁸ copies/mL in the untreated samples. Upon additionof antiviral compounds to the culture media, a dose-related decrease invirus production was observed. Maximal effect of suppression of viralTABLE 1 xxBRU 184V real-time real-time RT assay RT-PCR RT assay RT-PCREC₅₀ EC₉₀ EC₅₀ EC₉₀ EC₅₀ EC₉₀ EC₅₀ EC₉₀ AZT 0.0034 0.034 0.0039 0.0360.013 0.12 ND ND 3TC 0.018 0.077 0.03 0.12 >100 >100 ND ND d4T 0.00340.13 0.0027 0.12 0.032 0.19 0.00078 0.19 (−) FTC 0.011 0.05 0.0059 0.08865.1 160 0.34 >100 β-L-d4-2′F-(4-S-pentenyl)-C 1.61 11.6 0.42.68 >100 >100 0.091 34.9 β-D-d4-2′F-(4-S-pentenyl)-C 5.98 23.2 2.6818.1 >100 >100 11.8 >100 ND: not done

Example 15

MTS/MTT Toxicity and Real-Time PCR Mitochondrial DNA Polymerase Toxicity

Mitochondrial toxicity (γ-DNA polymerase inhibition) was evaluated byreal-time PCR, using the comparative cycle threshold (Ct) method.β-Actin served as an endogenous reference. All compounds were tested inroutine MTT or MTS toxicity assays (material and methods). In order tofind out whether these compounds should have any inhibitory effect onthe mitochondrial DNA polymerase γ, a real time PCR technology formitochondrial DNA polymerase toxicity was designed. In a first step,standard curves using 1-log diluted total HepG2 DNA were created, andshowed linearity over at least 4 logs (only 4-logs were tested for thesetargets). FIG. 4 demonstrates that efficiencies of target and referenceamplification are approximately equal, because the value of the slope ofinput amount versus ΔCt (Ct β-actin minus Ct mitochondrial, wherein Ctis the PCR cycle threshold where a sample becomes detectable) is lessthan 0.1.

Furthermore, total DNA was isolated from HepG2 cells cultured inpresence of the antiviral compound. The mitochondrial gene and theβ-actin gene were then amplified. There are at least two methods toobtain accurate quantity measurements, one method is using standardcurves, the other method is known as the comparative cycle thresholdmethod (User Bulletin # 2; Applied Biosystems, CA). Since both targets(mitochondrial and β-actin) are amplified with almost identicalefficiencies using the described primer-probe sets, either method can beused to measure the mitochondrial toxicities induced by antiviralagents. In our experiments, the comparative Ct method was used. Thismethod uses arithmetic formulas in which the amount of target(mitochondrial DNA) is normalized to an endogenous reference (β-actingene) and is relative to a calibrator (no drug control at day 7). Thisarithmetic formula is given by 2^(−ΔΔCt).

The relative mitochondrial DNA polymerase γ toxicity of two antiviralcompounds (−)-FTC and D-DDC were compared alongside. FIG. 5 demonstratesthe results obtained for each antiviral agent. It is clear from thisfigure that (−)-FTC does not induce any significant mitochondrial DNAreduction as compared to the no-drug control. Instead, importantdifferences were observed for D-DDC at 1 and 10 μM concentration. D-DDCdemonstrated dose-dependent reduction in mitochondrial DNA synthesis ascompared to the no-drug control. The β-L and β-D analogues of2′,3′-didehydro-2′,3′-dideoxy-2′-fluoro-4′-thio-cytidine[“d4-2′-F-(4S-pentenyl)-C”] both showed no toxicity after a 7-dayincubation with up to 10 μM of the compounds using this approach.Similarly, in an MTS-dye assay (Promega), no cytotoxicity was observedfor these compounds in human PBMC, Vero and CEM cells when evaluated upto 100 μM; its CC₅₀ values were higher than 100 μM on all cell-typestested (HepG2, VERO, PBMC, and CEM).

Example 16

Cell culture assays were used to determine the anti-Flaviviridaeactivity of unmodified or modified ribonucleosides.

(a) RNA Isolation and Quantitative RT-PCR Analysis

An effective process to quantify the viral load in a host, termedreal-time polymerase chain reaction (“RT-PCR”) is provided. The processinvolves using a quenched fluorescent probe molecule that can behybridized to viral DNA or RNA. Therefore, upon exonucleolyticdegradation, a detectable fluorescent signal can be monitored.Therefore, the RT-PCR amplified DNA or RNA is detected in real time bymonitoring the presence of fluorescence signals.

As one illustration of this method, in the case of BVDV in MDBK cells,in a first step, viral RNA is isolated from 140 μL of the cell culturesupernatant by means of a commercially available column (Viral RNAextraction kit, QiaGen, CA). The viral RNA is then eluted from thecolumn to yield a total volume of 60 μL, and subsequently amplified witha quantitative RT-PCR protocol using a suitable primer for the BVDV NADLstrain. A quenched fluorescent probe molecule is hybridized to the BVDVDNA, which then undergoes exonucleolytic degradation resulting in adetectable fluorescent signal. Therefore, the RT-PCR amplified DNA wasdetected in real time by monitoring the presence of fluorescencesignals. The TaqMan probe molecule(5′-6-FAM-AAATCCTCCTAACAAGCGGGTTCCAGG-TAMRA 3′ [Sequence ID No] andprimers (sense: 5′-AGCCTTCAGTTTCTTGCTGATGT-3′ [Sequence ID No]; andantisense: 5′-TGTTGCGAAAGCACCAACAG-3′ [Sequence ID No]) were designedwith the aid of the Primer Express software (PE-Applied Biosystems) tobe complementary to the BVDV NADL NS5B region. A total of 10 μL of RNAwas analyzed in a 50 μL RT-PCR mixture. Reagents and conditions used inquantitative PCR were purchased from PE-Applied Biosystems. The standardcurve that was created using the undiluted inoculum virus ranged from6000 plaque forming units (PFU to 0.6 PFU per RT-PCR mixture. A linearrange of over 4-logs was routinely obtained.

A comparable approach can be taken to measure the amount of otherFlaviviridae (more importantly HCV, YFV, Dengue, West Nile Virus andothers) in a clinical sample or in a tissue culture sample. For example,the combination of HCV RNA purification with real-time RT-PCR using thefollowing primers (5′-TTCCGCAGACCACTATGG-3′ [Sequence ID No.] and5′-AGCCATGGCGTTAGTATGAGTGT-3′ [Sequence ID No.]) and probe(5′-6-FAM-CCTCCAGGACCCCCCCTCCC-TAMRA-3′ [Sequence ID No.]) resulted in a7-log linear range of viral load detection.

(b) Cell/Viral Materials

One of the best characterized members of the Pestivirus genus is BVDV.BVDV and HCV share at least three common features, which are thefollowing: (1) they the both undergo IRES-mediated translation; (2) NS4Acofactor is required by their NS3 serine protease; and (3) they undergosimilar polyprotein processing within the non-structural region,especially at the NS5A and NS5B junction site.

The BVDV replication system was used for the discovery ofanti-Flaviviridae compounds. The compounds described herein are activeagainst Pestiviruses, Hepaciviruses and/or Flaviviruses.

Maldin-Darby bovine kidney (MDBK) cells were grown and maintained in amodified eagle medium (DMEM/F12; GibcoBRL), supplemented with 10% heatinactivated horse serum at 37° C. in a humidified, 5% CO₂, incubator.

Bovine viral diarrhea virus (BVDV), strain NADL, causes a cytopathogeniceffect (CPE) after infection of these cells.

(c) Antiviral Assay

MDBK-cells, grown in DMEM/F12-10% horse serum (HS), were isolated instandard techniques using trypsin-EDTA. Cells were seeded in a 96-wellplate at 5×10⁴ cells/well, with test compound (20 micromolar (μM)concentration) to give a total volume of 100 microliters (μL). After onehour, the media was removed and the cells were infected at amultiplicity of infection (MOI) of 0.02 or 0.002 in a total volume of 50μL for 45 minutes. Thereafter, the virus was removed and the cells werewashed twice with 100 μL of assay media. Finally, the infected cellswere incubated in a total volume of 100 μL containing the test compoundat 10, 40 or 100 μM concentration. After 22 hours, the cell supernatantwas collected by removing the cellular debris by low-speedcentrifugation, and subsequently tested for the presence of virus in aquantitative manner.

(d) Cytotoxicity Testing of Candidate Anti-Flaviviridae Compounds

The cytotoxicity testing as performed here is a standard technique.Briefly, cells are seeded in 96-well plates at various concentrations(dependent on cell type, duration of assay), typically at 5×10³ cellsper well, in the presence of increasing concentrations of the testcompound (0, 1, 3, 10, 33, and 100 μM). After a three day-incubation,cell viability and mitochondrial activity are measured by adding theMTS-dye (Promega), followed by a 3 hours incubation. Afterwards theplates containing the dye are read at 490 nm. Such methodologies arewell described and available from the manufacturer (Promega).

Example 17

The BVDV RT-PCR Quantification Standard Curve

The standard BVDV virus stock contained 2×10⁶ PFU/mL, as determined byroutine plaque assay (Mendez, E. et al. J. Virol. 1998, 72, 4737). ViralRNA was extracted from 140 μL of this inoculum material and eluted froma column using 60 μL of an elution buffer. This purified RNA materialthen was diluted stepwise from 10⁻¹ to 10⁻⁵. Using the real-time RT-PCRamplification technique, 10 μL of each dilution was tested. The resultsof this dilution series are plotted in FIG. 1, relating PFU toconcentration of standard. From this experiment, it is clear that thistechnology allows for reliable quantification over 4-logs of virus (from6000 to 0.6 PFU/input in amplification mix). The lower limit ofdetection in this experiment is 0.6 PFU or −0.22 log PFU. Therefore, thereal-time RT-PCR quantification values of test samples below thisdetection limit were considered non-reliable.

Example 18

The BVDV Replication Cycle in MDBK Cells

In order to measure the BVDV production in MDBK cells and to determinethe optimal harvesting time over a certain period of time, cells wereseeded at 5×10⁴ cells/well and infected either with MOI=0.02 orMOI=0.002. After infection, the inoculum was removed and the cells werewashed twice with culture medium. At different time points, the cellsupernatant was harvested; and, the amount of virus was measured andcompared to the original inoculum and the cell wash. At least 2wash-steps were needed to remove the inoculum virus, as shown in FIG. 2.The amount of virus produced 22 hours after infection approximatelyequals the amount of virus used to inoculate the cells. Based on theseresults, the time required for one replication cycle of BVDV in MDBKcells was 22 hours. Note that the detection level set in theseexperiments was based on the lower limit of detection as determined bythe standard curve.

Example 19

Evaluation of Candidate Antiviral Compounds Using RT-PCR

MDBK cells were seeded at 5×10⁴ cells/well, infected with BVDV with amultiplicity of infection (MOI) equal to 0.02 and grown for 22 hours inthe presence of a test compound. Cells that were not treated with a testcompound were considered a negative control, while ribavirin served as apositive control. Viral RNA was extracted and analyzed by real timeRT-PCR. A typical experiment, shown in FIG. 3, demonstrates that thenegative control and the majority of the treated cells producedcomparable amounts of virus (between 1.5 and 2 log PFU/input),effectively showing the test compounds as non-active. However, the cellstreated with the positive control, ribavirin (RIB) or with5-hydroxyuridine (I-a-45) show an almost complete absence of viral RNA.RIB and I-a-45 reduce viral production by approximately 2 log PFU, or99%, in the 22 hour reproduction period. The exact potency of thesecompounds cannot be deduced from this kind of experiment, since thedetection limit in this experiment is set at −0.22 log PFU and only onecycle of viral replication occurs under the stated experimentalconditions.

Potencies, or the effect concentration of compounds that inhibits virusproduction by 50% or 90% (EC₅₀ or EC₉₀ values, respectively), ofanti-BVDV compounds were determined in a similar set of experiments, butover a broad range of test compound concentrations (0, 1, 3, 10, 33, 100μM). The EC₉₀ value refers to the concentration necessary to obtain a1-log reduction in viral production within a 22 hour period.

The invention has been described with reference to various specific andpreferred embodiments and techniques. However, it should be understoodthat many variations and modifications will be obvious to those skilledin the art from the foregoing detailed description of the invention andmay be made while remaining within the spirit and scope of theinvention.

1-32. (canceled)
 33. A kit for assessing mitochondrial toxicity of acompound, comprising a mixture of oligonucleotides comprising at leastone first primer that provides a detectable signal on the occurrence ofamplification of mitochondrial nucleic acid; and at least one secondprimer that provides a second detectable signal on the occurrence ofamplification of host nuclear nucleic acid.
 34. A kit as in claim 33wherein the second primer comprises SEQ ID No. 1 and SEQ ID No.
 2. 35. Akit as in claim 34 further comprising SEQ ID No.
 3. 36. A kit as inclaim 35 further comprising a fluorescent dye and a quenching dye.
 37. Akit as in claim 33 wherein the first primer comprises SEQ ID No.
 19. 38.A kit as in claim 37 further comprising a third primer that comprisesSEQ ID No.
 20. 39. A kit as in claim 38 further comprising a sixthprimer that comprises SEQ ID No.
 21. 40. A kit as in claim 39 furthercomprising a fluorescent dye and a quenching dye.